Antipathogenic synthetic peptides and compositions comprising them

ABSTRACT

Non-hemolytic cytolytic agents selected from peptides, complexes of bundled peptides, mixtures of peptides or random peptide copolymers have a selected cytolytic activity manifested in that they have a cytolytic activity on pathogenic cells, being cells which are non-naturally occurring with the body consisting of microbial pathogenic organisms and malignant cells; and are non-hemolytic, having no cytolytic effect on red blood cells. The peptides may be cyclic derivatives of natural peptides such as pardaxin and mellitin and fragments thereof in which L-amino acid residues are replaced by corresponding D-amino acid residues, or are diastereomers of linear peptides composed of varying ratios of at least positively charged amino acid and at least one hydrophobic amino acid, and in which at one of the amino acid residues is a D-amino acid. Pharmaceutical compositions comprising the non-hemolytic cytolytic agents can be used for the treatment of several diseases caused by pathogens including antibacterial, fungal, viral mycoplamsa and protozoan infections and for the treatment of cancer.

CROSS REFERENCE TO RELATED APPLICATION

The present application is the national stage under 35 U.S.C. 371 ofPCT/IL98/00081, filed Feb. 19, 1998 which claims priority to applicationPCT/IL97/00066, FILED Feb. 20, 1997.

FIELD OF THE INVENTION

The present invention concerns novel non-hemolytic cytolytic agents,compositions comprising them and their use in the treatment of diseasesor disorders and in agriculture.

BACKGROUND OF THE INVENTION

In the text below, reference is being made to prior art documents, thecomplete particulars of which can be found in the “References” sectionat the end of the specification before the claims.

The increasing resistance of microorganisms to the availableantimicrobial drugs has resulted in extensive studies focused ondeveloping alternative antimicrobial compounds.

In addition, or complementary, to the highly specific cell-mediatedimmune response, vertebrates and other organisms have a defense systemmade up of distinct groups of broad spectrum cytolytic, e.g.,antibacterial, peptides.

Studies on lipid-peptide interactions of such cytolytic peptides, alsoknown as cytolysins, tend to emphasize the importance of the amphipathicα-helical structure for their cytolytic activity. This conclusion isbased mainly on studies with cytolysins that act on either mammaliancells or bacteria alone or on both types of cells. A major group ofcytolytic peptides in this family are host-defense short linear peptides(≦40 amino acids), which are devoid of disulfide bridges (Boman, 1995).The peptides vary considerably in chain length, hydrophobicity andoverall distribution of charges, but share a common structure uponassociation with lipid bilayers, namely, an amphipathic α-helixstructure (Segrest et al., 1990).

Examples of known cytolysins include: (i) antibacterial peptides thatare cytolytic to bacteria only, e.g. cecropins, isolated from thececropia moth (Steiner et al., 1981), magainins (Zasloff, 1987) anddermaseptins (Mor et al., 1991) isolated from the skin of frogs; (ii)cytolysins that are selectively cytotoxic to mammalian cells, such asδ-hemolysin isolated from Staphylococcus aureus (Dhople and Nagaraj,1993); and (iii) cytolysins that are not cell-selective, such as the beevenom melittin (Habermann and Jentsch, 1967) and the neurotoxin pardaxin(Shai et al., 1988) that lyse both mammalian cells and bacteria.

Antibacterial peptides were initially discovered in invertebrates, andsubsequently in vertebrates, including humans. As a complementary oradditional defense system, this secondary, chemical immune systemprovides organisms with a repertoire of small peptides that aresynthesized promptly upon induction, and which act against invasion byoccasional and obligate pathogens as well as against the uncontrolledproliferation of commensal microorganisms (Boman, 1995). So far, morethan 100 different antibacterial peptides have been isolated andcharacterized. The largest family, and probably the most studied,includes those peptides that are positively charged and adopt anamphipathic α-helical structure. Numerous studies conducted on variousnative antibacterial peptides tend to emphasize the importance of anamphipathic α-helical structure and a net positive charge for cytolyticactivity. The positive charge facilitates interaction of the peptideswith the negatively-charged membranes (Andreu et al., 1985) found inhigher concentrations in the pathogenic cell membrane as compared tonormal eukaryotic cells, and the amphipathic α-helical structure isessential for lytic activity (Chen et al., 1998). Such interactions havebeen proposed to destroy the energy rhetabolism of the target organismby increasing the permeability of energy-transducing membranes (Okadaand Natori, 1984). Because of their amphipathic structure, it has beensuggested that these antibacterial peptides permeate the membrane byforming ion channels/pores via a “barrel-stave” mechanism (Rizzo et al.,1987). According to this model transmembrane amiphiphilic α-helices formbundles in which outwardly-directed hydrophobic surfaces interact withthe lipid constituents of the membrane, while inwardly facinghydrophilic surfaces produce a pore. Alternatively, the peptides bindparallel to the surface of the membrane, cover the surface of themembrane in a “carpet”-like manner and dissolve it like a detergent(Shai, 1995).

Despite extensive studies, the exact mode of action of short linear noncell-selective peptides, such as pardaxin and melittin, is not knownyet, and it is not clear whether similar structural features arerequired for their cytotoxicity towards mammalian cells and bacteria.

Pardaxin, a 33-mer peptide, is an excitatory neurotoxin that has beenpurified from the Red Sea Moses Sole Pardachirus marmoratus (Shai etal., 1988) and from the Peacock Sole of the western Pacific Pardachiruspavoninus (Thompson et al., 1986). Pardaxin possesses a variety ofbiological activities depending upon its concentration (reviewed inShai, 1994). At concentrations below 10⁻⁷ M, pardaxin induces therelease of neurotransmitters in a calcium-dependent manner. At higherconcentrations of 10⁻⁷ M to 10⁻⁵M, the process is calcium-independent,and above 10⁻⁵M cytolysis is induced. Pardaxin also affects theactivities of various physiological preparations in vitro. Itsbiological roles have been attributed to its interference with the ionictransport of the osmoregulatory system in epithelium and to presynapticactivity by forming ion channels that are voltage dependent and slightlyselective to cations. A “barrel-stave” mechanism for insertion ofpardaxin into membranes was proposed on the basis of its structure andvarious biophysical studies (reviewed in Shai, 1994). Pardaxin has ahelix-hinge-helix structure: the N-helix includes residues 1–11 and theC-helix includes residues 14–26. The helices are separated by a prolineresidue situated at position 13. This structural motif is found both inantibacterial peptides that can act specifically on bacteria (e.g.,cecropin), and in cytotoxic peptides that can lyse a variety of cells(e.g., melittin).

Melittin, a 26-mer amphipathic peptide, is the major component of thevenom of the honey bee Apis mellifera (Habermann and Jentsch, 1967) andis one of the most studied membrane-seeking peptides (Dempsey, 1990).Melittin is highly cytotoxic for mammalian cells, but is also a highlypotent antibacterial agent (Steiner et al., 981). Numerous studies havebeen undertaken to determine the nature of the interaction of melittinwith membranes, both with the aim of understanding the molecularmechanism of melittin-induced hemolysis and as a model for studying thegeneral features of structures of membrane proteins and interactions ofsuch proteins with phospholipid membranes. Much of the currentlydescribed evidence indicates that different molecular mechanisms mayunderlie different actions of melittin. Nevertheless, the amphipathicα-helical structure has been shown to be a prerequisite for its variousactivities (Perez et al., 1994).

The structure of melittin has been investigated using varioustechniques. The results of X-ray crystallography and NMR in methanolicsolutions indicate that the molecule consists of two α-helical segments(residues 1–10 and 13–26) that intersect at an angle of 120°. Thesesegments are connected by a hinge (11–12) to form a bent α-helical rodwith the hydrophilic and hydrophobic sides facing opposite directions.Four such monomeric melittin molecules cluster together, throughhydrophobic interactions, to form a tetramer (Anderson et al., 1980;Bazzo et al., 1998; Terwilliger and Eisenberg, 1982; Terwilliger andEisenberg, 1982). Upon initial interaction with membrane surfaces, ithas been found that the tetramer dissociates to monomers, which retainα-helical conformation prior to insertion into the membrane (Altenbachand Hubbell, 1988).

Melittin shares some similarities with pardaxin. Both pardaxin andmelittin are composed of two helices with a proline hinge between them.Furthermore, they exhibit significant homology in their N-helices, whichare mostly hydrophobic (Thompson et al., 1986). However, pardaxin (netcharge +1) contains an additional seven amino acids residue at itsC-terminal side with a charge of −2, while melittin (net charge α6)terminates with an amide group and contains the positively-chargedtetrapeptide sequence Lys-Arg-Lys-Arg. There are several functionaldifferences between pardaxin and melittin. Pardaxin binds similarly toboth zwitterionic and negatively charged phospholipids (Rapaport andShai, 1991), while melittin binds better to negatively charged than tozwitterionic phospholipids (Batenburg et al., 1987; Batenburg et al.,1987). Also, pardaxin binds to phospholipids with positive cooperativity(Rapaport and Shai, 1991) while melittin binds with negativecooperativity (Batenburg et al., 1987; Batenburg et al., 1987). Althoughboth pardaxin and melittin are potent antibacterial peptides againstGram-positive and Gram-negative bacteria, pardaxin is 40–100 fold lesshemolytic than melittin towards human erythrocytes (Oren and Shai,1996).

Analogues of pardaxin with L- to D-substitutions were shown to becapable of lysing human erythrocytes (Pouny and Shai, 1992). It waslater shown (see results reported below) that two of the peptidesdisclosed in Pouny and Shai, 1992, namely, D-Pro⁷-pardaxin andD-Leu¹⁸Leu¹⁹-pardaxin, while being hemolytic, have a very lowantibacterial activity. Analogues of magainin with L- to D-substitutionswere also found to lack antibacterial activity (Chen et al., 1988).

GLOSSARY

In the following, use will be made of several coined terms for thepurpose of streamlining reading of the text and facilitating betterunderstanding of the invention. it should be noted, however, that forcomplete understanding of these terms, reference will at times be madeto the complete description below. These terms and their meaning hereinare the following:

“Heterogeneous peptide” as used herein refers to a peptide comprisingboth D- and L-amino acid residues.

“Homogeneous peptide” as used herein refers to a peptide comprisingeither only the natural L-amino acid residues, or only D-amino acidresidues.

“Homogeneous L-peptide” and “homogeneous D-peptide” as used hereinrefers the homogeneous polypeptide consisting entirely of either L- orD-amino acid residues, respectively.

“Heterogeneous L-based peptide” and “heterogeneous D-based peptide” asused herein refers to a heterogeneous peptide comprising primarilyL-amino acid residues, e.g., a peptide derived from homogeneousL-peptide in which one or more of the L-amino acid residues has beenreplaced by counterpart D-enantiomers, and a heterogeneous peptidecomprising primarily D-amino acid residues in which one or more of theD-amino acid residues has been replaced by counterpart L-enantiomers,respectively.

“Helical peptide” as used herein refers to a peptide having a continuousα-helix stretch throughout the major portion of its length. The helicalportion of a helical peptide consists entirely of either L-amino acidresidues or D-amino acid residues.

“Non-helical peptide” as used herein refers to a peptide which has noα-helix structure or has non-continuous α-helix structures dispersedalong its length. A non-helical peptide according to the invention mayhave an α-helix stretch which, in case it is terminal, has a lengthspanning less than half a width of a cell's membrane, e.g., less thanabout 10–15 amino acid residues, and if it is a non-terminal α-helix,has a length which is less than the full width of the cell's membrane,e.g., less than about 20–25 amino acid residues. A non-helical peptidemay be a homogeneous peptide with α-helix breaker moieties (see below)or may be a heterogeneous peptide.

“α-helix breaker moiety” as used herein refers to a moiety which ifinserted into an α-helix structure disrupts its continuity. Such amoiety may for example be the amino acid residue proline or glycine,α-methyl-substituted α-amino acids, non-α-amino acids both cyclic andacyclic such as 6-amino-hexanoic acid, 3-amino-1-cyclohexanoic acid,4-amino-1-cyclohexanoic acid or may be an L- or D-enantiomer insertedinto an α-helix stretch consisting of a stretch of amino acid residuesof the opposite enantiomer.

“Pathogenic cells” as used herein refers to cells which arenon-naturally occurring within the body, including cancer cells andpathogenic organisms such as bacteria, fungi, protozoa, virus andmycoplasma, as well as mammalian cells infected with pathogenicorganisms such as parasitic protozoans, e.g Leishmania and Plasmodium.

“Selective cytolytic activity” as used herein refers to activity of anagent in inducing cytolysis of a pathogenic cell, the selectivity beingmanifested in that the agent induces cytolysis of the pathogenic cellsat a much lower concentration to that required for the cytolysis ofnormal non-pathogenic cells such as red blood cells.

“Non-hemolytic” as used herein refers to agents which cause hemolysis ofred blood cells at much higher concentrations than the concentrationrequired to cause cytolysis of other cells, such as pathogenic cellssuch as microorganism cells, cancer cells, and the like.

“Diastereomers” is used herein as a synonym of “heterogeneous peptide”.

SUMMARY OF THE INVENTION

The present invention provides a non-hemolytic cytolytic agent selectedfrom a peptide, a complex of bundled peptides, a mixture of peptides ora random peptide copolymer, said agent having a selective cytolyticactivity manifested in that it has a cytolytic activity on pathogeniccells; being cells which are non-naturally occurring within the bodyconsisting of microbial pathogenic organisms and malignant cells; and itis non-hemolytic, namely it has no cytolytic effect on red blood cellsor has a cytolytic effect on red blood cells at concentrations which aresubstantially higher than that in which it manifests said cytolyticactivity, said non-hemolytic cytolytic agent being selected from thegroup consisting of:

-   -   (1) a cyclic derivative of a peptide having a net positive        charge which is greater than +1, and comprising both L-amino        acid residues and D-amino acid residues, or comprising one or        both of L-amino acid residues and D-amino acid residues, and        comprising an α-helix breaker moiety;    -   (2) a peptide comprising both L-amino acid residues and D-amino        acid residues, having a net positive charge which is greater        than +1, and having a sequence of amino acids such that a        corresponding amino acid sequence comprising only L-amino acid        residues is not found in nature, and cyclic derivatives thereof;    -   (3) a complex consisting of a plurality of 2 or more        non-hemolytic cytolytic peptides, each peptide having a net        positive charge which is greater than +1, and comprising both        L-amino acid residues and D-amino acid residues, or comprising        one or both of L-amino acid residues and D-amino acid residues        and comprising an α-helix breaker moiety, or cyclic derivatives        of the foregoing, said peptides being bundled together by the        use of a linker molecule covalently bound to each of the        peptides;    -   (4) a mixture consisting of a plurality of 2 or more        non-hemolytic cytolytic peptides, each peptide having a net        positive charge which is greater than +1, and comprising both        L-amino acid residues and D-amino acid residues, or comprising        one or both of L-amino acid residues and D-amino acid residues        and comprising an α-helix breaker moiety, or cyclic derivatives        of the foregoing; and    -   (5) a random copolymer consisting of different ratios of a        hydrophobic, a positively charged and a D-amino acid.

In one embodiment, the cyclic derivatives of (1) above are derived froma non-selective cytolytic natural peptide such as for example pardaxinand mellitin or from a fragment thereof. These cyclic diastereomers areobtained by conventional cyclization methods for peptides. In oneembodiment, the cyclic diastereomer is derived from the fragment 1–22 ofpardaxin to which 1 to 3 Lys residues have been added to the N-terminusand cysteine residues have been added to both N- and C-terminus forcyclization.

The net positive charge of the peptides may be due to the native aminoacid composition, to neutralization of free carboxyl groups, and/or tothe addition of positively charged amino acid residues or positivelycharged chemical groups.

In another embodiment, the invention provides a non-hemolytic cytolyticpeptide and cyclic derivatives thereof as defined in (2) above, havingthe following characteristics:

-   -   (a) it is a non-natural synthetic peptide composed of varying        ratios of at least one hydrophobic amino acid and at least one        positively charged amino acid, and in which sequence at least        one of the amino acid residues is a D-amino acid;    -   (b) the peptide has a net positive charge which is greater than        +1; and    -   (c) the ratio of hydrophobic to positively charged amino acids        is such that the peptide is cytolytic to pathogenic cells but        does not cause cytolysis of red blood cells.

Examples of positively charged amino acids are lysine, arginine andhistidine, and of hydrophobic amino acids are leucine, isoleucine,glycine, alanine, valine, phenylalanine, proline, tyrosine andtryptophan. The net positive charge is due to the amino acidcomposition, but the addition of positively charged chemical groups mayalso be considered. In addition, polar amino acids such as serine,threonine, methionine, asparagine, glutamine and cysteine, may be addedin order to decrease the hydrophobicity and/or the toxicity of themolecule. In one preferred embodiment, the peptide is composed of onehydrophobic amino acid such as leucine, alanine or valine, and onepositively charged amino acid such as lysine or arginine. The syntheticnon-natural peptide may have at least 6, particularly ten or more aminoamino acid residues. In one preferred embodiment, the syntheticdiastereomer is a 12-mer peptide composed of leucine, alanine or valineand lysine, and at least one third of the sequence is composed ofD-amino acids.

In still another embodiment, the invention provides a non-hemolyticcytolytic complex as defined in (3) above, consisting of a plurality of2 or more non-hemolytic cytolytic peptides complexed or “bundled”together, e.g. by the use of a linker or “template” molecule covalentlybound to each of the peptides. The bundle may be composed of 2 or more,preferably 5, molecules of the same peptide or of different peptides.The linker/template may be a peptide or a commonly used linker, e.g.polymers such as polyesters, polyamides, polypeptides, polyaminoacids(e.g. polylysine) carrying active groups such as OH, SH, COOH, NH₂,CH₂Br.

In still a further embodiment, the invention provides a non-hemolyticcytolytic mixture as defined in (4) above, obtained by adding a mixturecomposed of 1 eq each of the desired hydrophobic, positively charged andD-amino acid at each coupling step of the solid phase method for peptidesynthesis. In this way, a mixture of 3¹² different peptides wereobtained with a mixture of lysine, leucine and D-leucine, and themixture was obtained therefrom after HF cleavage, extraction with waterand lyophilization.

In a further embodiment, the invention provides a non-hemolyticcytolytic random copolymer as defined in (5) above consisting ofdifferent ratios of a hydrophobic, a positively charged and a D-aminoacid, e.g. 1:1:1, 2:1:1 and 3:1:1 (Mol) copolymers of Lys:Leu:D-Leu.

Preferably, the non-hemolytic cytolytic peptide has either no α-helixstructure or has an α-helix structure which length is insufficient tospan the width of a cell membrane. The peptide thus does not contain anuninterrupted stretch of either all D- or all L-amino acid residues of alength capable of forming part of a transmembrane pore. Such a length istypically about 20–22 amino acids, where the stretch is in thenon-terminal portion of the peptide and about half, i.e., 10–11 aminoacids, where the stretch is in the terminus of the peptides, since insuch a case two peptides may join their terminus together and span thecell's membrane.

The disruption of a stretch of D- or L-amino acids residues may becarried out by replacement of one or more amino acids in the stretch bythe amino acid of the opposite enantiomer or by placing in thecontinuous stretch an α-helix breaker moiety such as proline, glycine,an α-methyl-α-amino acid or a non-α-amino acid.

The peptides of the invention and the peptides comprised within thecomplexes, mixtures and copolymers of the invention have a net positivecharge greater than +1. The net positive charge may be due to the nativeamino acid composition of the invention, to neutralization of free COOHgroups, for example by amidation, or may be due to addition ofpositively charged amino acids or chemical groups. It was found that theselective cytolytic activity can at times be enhanced by increasing thenet positive charge, for example, by attaching at any position in themolecule a positively charged amino acid and/or a positively chargedgroup. For example, a polyamine group, an alkylamino group or aminoalkylamino group, etc., may be attached at one of its terminals,typically at its carboxyl terminal. A preferred such group is theaminoethylamino group —NH—CH₂—CH₂—NH₂, designated hereinafter “TA”.

The peptides that are derived from non-selective cytolytic naturalpeptides, e.g. pardaxin and melittin, are amphipathic, meaning that theyhave one surface which is mainly composed of hydrophobic amino acidresidues and an opposite surface which is mainly composed of hydrophilicamino acid residues. The amphipathic nature of peptides may be verifiedaccording to methods known in the art. An example of such a method isthe use of a Shiffer and Edmondson wheel projection wherein the aminoacid residues are written, according to their sequence in a circle sothat each amino acid in the sequence is angularly displaced by 100° fromits neighboring amino acid residues (3.6 amino acids per circle). Ifmost hydrophilic amino acids concentrate on one side of the wheel andhydrophobic amino acids concentrate on the opposite side of the wheelthen the peptide may be considered amphipathic.

The peptides of the invention that are not derived from non-selectivecytolytic natural peptides, e.g. the synthetic diastereomers composed ofhydrophobic, positively charged and D-amino acids, are not amphipathic.They have a net positive charge greater than +1 and a suitablehydrophobic to positively charged amino acid ratio such that theresulting peptide is cytolytic to pathogenic cells but not hemolytic.These peptides can be screened very easily according to the invention byusing the antibacterial and hemolytic tests described herein. In oneembodiment, for a peptide composed of leucine and lysine, an appropriateLeu:Lys ratio may be 64%:36% for a diastereomer of 6 amino acidresidues, and 66%:34% for a diastereomer of 12 amino acid residues.

Without wishing to be bound by theory, it is believed however that thecytolytic activity may be the result of aggregation of a number ofpeptides on the surface of the membrane and together such peptides causelesion of the cell membrane. Accordingly, as described above, it iscontemplated in accordance with the invention also to use a plurality ofpeptides either as a mixture or complexed (or bundled) together, e.g.,by the use of a linker molecule covalently bound to each of thepeptides.

The individual peptide typically consists of at least six, andpreferably ten or more amino acid residues. In a complex of theinvention, each individual peptide may typically have a length of above5 amino acid residues.

The present invention also provides a pharmaceutical compositioncomprising a non-hemolytic cytolytic agent of the invention as theactive ingredient, and a pharmaceutically acceptable carrier. Thecompositions are for use in the treatment of diseases or disorderscaused by different pathogenic organisms such as Gram-positive andGram-negative bacteria, virus, fungi, mycoplasma, and parasiticprotozoa, e.g Leishmania that causes leishmaniasis and Plasmodium thatcauses malaria. In a preferred embodiment, the anti-pathogeniccomposition is an antimicrobial, particularly antibacterialcompositions. In addition, the compositions of the invention are usefulagainst malignant cells and can be used in the treatment of cancer.

Also provided by the present invention is a method of treatmentcomprising administering said hemolytic non-cytolytic agent to a subjectin need. The method of the invention as well as the above compositionare applicable in both human and veterinary medicine.

Further provided in accordance with the invention is also the use ofsaid non-hemolytic cytolytic agent in the preparation of apharmaceutical composition for the treatment of a disease or a disorderin human or a non-human animal, in particular antibacterialcompositions.

In a further embodiment, the selective agents of the invention can beused as disinfectants for the destruction of microorganisms, i.e., insolution for wetting contact lenses, may be used as preservatives, forexample in the cosmetic or food industry, and as pesticides, e.g.fungicides, bactericides, in agriculture, or for preservation ofagricultural products, e.g. fruits and legumes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows circular dichroism (CD) spectra of aminoethylaminopardaxin(TApar)-derived peptides. Spectra were taken at peptide concentrationsof 0.8–2.0×10⁻⁵ M in 40% 2,2,2-trifluoroethanol (TFE)/water. Symbols:TApar (−); [D]P⁷-TApar (.....); [D]L¹⁸L¹⁹-TApar (−) and[D]P⁷L¹⁸L¹⁹-TApar (·—·—);

FIG. 2 depicts dose-response curves of the hemolytic activity ofTApar-derived peptides towards human red blood cells (hRBC). The insetshows the assay results at low concentration. Symbol: Filled squares,melittin; filled triangles, TApar; filled circles, [D]P⁷-TApar; emptycircles, [D]L¹⁸L¹⁹-TApar; empty squares, [D]P⁷L¹⁸L¹⁹-TApar; emptytriangles, dermaseptin.

FIGS. 3A–B show the maximal dissipation of the diffusion potential invesicles induced by the TApar-derived peptides. The peptides were addedto isotonic K⁺ free buffer containing small unilamellar vesicles (SUV)composed of egg phosphatidylcholine/phosphatidylserine (PC/PS) (FIG. 3A)or PC (FIG. 3B), pre-equilibrated with the fluorescent dye diS-C₂-5 andvalinomycin. Fluorescence recovery was measured 10–20 min after thepeptides were mixed with the vesicles. Symbols: Filled triangles, TApar;filled circles, [D]P⁷-TApar; empty circles, [D]L¹⁸L¹⁹-TApar; emptysquares, [D]⁷L¹⁸L¹⁹-TApar;

FIGS. 4A–C show electron micrographs of negatively stained E.coli celluntreated (FIG. 4A) or treated with [D]P⁷L¹⁸L¹⁹-TApar at concentrationlower than the minimal inhibitory concentration (MIC) (4B) or at MICconcentrations (4C);

FIG. 5 shows CD spectra of melittin and melittin-derived diastereomers.Spectra were taken at peptide concentrations of 0.8–0.2×10⁻⁵ M in 40%TFE/water. Symbols: melittin, (_); [D]-V^(5,8)I¹⁷,K²¹-melittin, (. . . ..); [D]-V^(5,8),I¹⁷,K²¹-melittin-COOH, (- - - - -).

FIG. 6 depicts dose-response curves of the hemolytic activity of themelittin-derived diastereomers towards hRBC. Symbols: filled circles,melittin; empty circles, [D]-V^(5,8),I¹⁷,K²¹-melittin-COOH; filledtriangles, [D]-V^(5,8),I¹⁷,K²¹-melittin.

FIGS. 7A–C shows electron micrographs of negatively stained E. coliuntreated (FIG. 7A) or treated with [D]-V^(5,8),I¹⁷,K²¹-melittin atconcentrations lower than the MIC (7B) or at the MIC concentrations(7C).

FIGS. 8A–B shows maximal dissipation of the diffusion potential invesicles induced by melittin and a melittin-derived diastereomer. Thepeptides were added to isotonic K⁺ free buffer containing SUV composedof PC (8A) or PC/PS (8B), pre-equilibrated with the fluorescent dyediS-C₂-5 and valinomycin. Fluorescence recovery was measured 10–20 minafter the peptides were mixed with the vesicles. Symbols: filledcircles, melittin; filled triangles, [D]-V^(5,8),I¹⁷,K²¹-melittin.

FIGS. 9A–B show increase in the fluorescence of[D]-V^(5,8),I¹⁷,K²¹-melittin (0.5 μM total concentration) upon titrationwith PC/PS vesicles (filled triangles) or PC vesicles (empty triangles),with excitation wavelength set at 280 nm and emission at 340 nm. Theexperiment was performed at 25° C. in 50 mM Na₂SO₄, 25 mM HEPES-SO₄ ⁻²pH 6.8 (FIG. 9A); and binding isotherm derived from FIG. 9A by plottingX_(b)* (molar ratio of bound peptide per 60% of lipid) versus C_(f)(equilibrium concentration of free peptide in the solution) (FIG. 9B).

FIGS. 10A–B show quenching of the environmentally sensitive tryptophanby brominated phospholipids. Melittin (FIG. 10A) and[D]-V^(5,8),I¹⁷,K²¹-melittin (10B) were added to buffer containing PC/PS(1:1 w/w) SUV. The SUV contained 25% of either 6,7 Br-PC (_·_·_·_·), or9,10 Br-PC (- - - - -), or 11,12 (Br-PC) (· · · · ·). After 2 minincubation, an emission spectrum of the tryptophan was recorded usingspectrofluorometer with excitation set at 280 nm. For comparison PC/PS(1:1 w/w) SUV with no Br-PC were used (_).

FIG. 11 shows the effect of the hydrophobicity of the Leu/Lysdiastereomers on RP-HPLC retention time.

FIG. 12 shows dose-response curves of the hemolytic activity of theLeu/Lys diastereomers towards hRBC. The inset shows the assay results atlow concentrations. Symbols: empty squares, melittin; filled squares,[D]-L^(3,4,8,10)-K₃L₉; filled circles, [D]-L^(3,4,8,10)-K₄L₈; emptytriangles, [D]-L^(3,4,8,10)-K₅L₇; filled triangles,[D]-L^(3,4,8,10)-K₇L₅.

FIGS. 13A–B show maximal dissipation of the diffusion potential invesicles, induced by the Leu/Lys diastereomers. The peptides were addedto isotonic K⁺ free buffer containing SUV composed of PC (FIG. 13A) orPE/PG (13B); pre-equilibrated with the fluorescent dye diS-C₂-5 andvalinomycin. Fluorescence recovery was measured 3–10 min after thepeptides were mixed with the vesicles. Symbols: filled squares,[D]-L^(3,4,8,10)-K₃L₉; filled circles, [D]-L^(3,4,8,10)-K₄L₈; filledtriangles, [D]-L^(3,4,8,10)-K₅L₇; crossed circles,[D]-L^(3,4,8,10)-K₇L₅.

FIGS. 14A–H show electron micrographs of negatively stained E. coliuntreated and treated with the various Leu/Lys diastereomers at 80% oftheir MIC. FIG. 14A, control; FIG. 14B, E. coli treated with[D]-L^(3,4,8,10)-K₃L₉; FIG. 14C, E. coli treated with[D]-L^(3,4,8,10)-K₄L₈; FIG. 14D, E. coli treated with[D]-L^(3,4,8,10)-K₅L₇; FIG. 14E, E. coli treated with[D]-L^(3,4,8,10)-K₇L₅; FIG. 14F, control; FIG. 14G, E. coli treated with[D]-L^(3,4,8,10)-K₄L₈; FIG. 14H, E. coli treated with[D]-L^(3,4,8,10)-K₅L₇.

DETAILED DESCRIPTION OF THE INVENTION

Heterogeneous L-based peptides have been found by the present inventorsto possess selective cytolytic activity manifested by a selectivedestruction of pathogenic cells, e.g., bacteria, with little or noeffect on non-pathogenic cells, i.e., red blood cells. This finding isvery surprising in view of the prevalent belief in the art that thecytolytic activity of cytolytic peptides in cells, whether pathogeniccells such as bacteria or normal mammalian cells, arises from a singleunderlying mechanism associated with the α-helix configuration.

Functional and structural studies with D-amino acid incorporatedanalogues (diastereomers) of pardaxin and melittin, two known non-cellselective cytolysins, carried in order to understand the molecularmechanism underlying cell selectively, revealed that the resultingdiastereomers did not retain their α-helical structure, which causedabrogation of their cytotoxic effects on mammalian cells. However, thediastereomers retained a high antibacterial activity, which wasexpressed by complete lysis of both Gram-positive and Gram-negativebacteria. Thus, the α-helical structure of pardaxin and melittin wasshown to be important for cytotoxicity against mammalian cells, but notto be a prerequisite for antibacterial activity. However, in anotherstudy, a single D-amino acid incorporated into the non-hemolyticantibacterial peptide magainin abolished almost totally itsantibacterial activity (Chen et al., 1988). The results with pardaxinand melittin diastereomers suggest that hydrophobicity and a netpositive charge confer selective antibacterial activity to non-selectivecytolytic peptides and that amphipathic α-helical structure is notrequired. However, the diastereomers of pardaxin and melittin containedlong stretches of L-amino acids (14–17 aa long) which raises thepossibility that the low residual helicity could be sufficient formembrane binding and destabilization.

To examine whether modulating hydrophobicity and the net positive chargeof linear cytotoxic peptides is sufficient to confer selectiveantibacterial activity, we chose to investigate diastereomers of shortmodel peptides (12 aa. long), composed of varying ratios of leucine andlysine and one third of their sequence composed of D-amino acids.Peptide length and the position of D-amino acids were such that shortpeptides with very short consecutive stretches of 1–3 L-amino acids thatcannot form an α-helical structure were constructed. The diastereomerswere evaluated with regard to (1) their cytotoxicity against bacteriaand human erythrocytes, (2) their structure, and (3) their ability tointeract and perturb the morphology of the bacterial wall and modelphospholipid membranes. The data show that modulating hydrophobicity andpositive charge is sufficient to confer antibacterial activity andcytolytic selectivity. Furthermore, the resulting antibacterial peptidesact synergistically at non lethal concentrations with availableantibacterial drugs such as tetracycline, and they are totally resistantto human serum inactivation which dramatically reduces the activity ofnative antibacterial peptides. Further shorter diastereomers (66 aa and8 aa long) were prepared and tested and found to be non-hemolyticcytolytic.

The finding that certain cytolytic non-helical peptides have ananti-pathogenic activity, paves the way for the preparation ofanti-pathogenic agents, which comprise such non-helical polypeptides.Where the non-helical peptides are heterogeneous peptides composed ofboth L-amino acids and D-amino acids, the anti-pathogenic agents havethe additional advantage of being more resistant to degradation, forexample by proteases, than homologous L-peptides, on the one hand, andon the other hand, are not completely degradation-resistant as the fullhomologous D-peptides. Resistance to degradation may be disadvantageousin view of slow clearance from the body with possible associated toxicside effects. The non-α-helical antipathogenic peptides may be used in avariety of therapeutic procedures.

Since it is known that homologous D-peptides possess essentiallyidentical cytolytic activity to the corresponding homologous L-peptides(Bessalle et al., 1990) then accordingly it is clear that heterogeneousD-based peptides possess the same antipathogenic properties asheterogeneous L-based peptides.

The finding that certain non-α-helical peptides have a cytolyticactivity against bacteria without a cytolytic activity against red bloodcells, is a result of the fact that bacterial cells differ from redblood cells in the composition of their cell membrane. Differences inthe compositions of the cell membrane can also be found among a varietyof pathogenic cells, such as cancer cells, and normal cells. Thus, basedon this finding, the agents of the present invention pave the way fordevelopment of a variety of drugs having a selective cytolytic activityagainst one class of cells within the body such as bacteria cells, cellsof a parasite, fungus cells, protozoa cells, or cancer cells, withlittle or no activity against non-pathogenic normal body cells.

The non-hemolytic cytolytic agents the invention having a selectivecytolytic activity against pathogenic cells, while having a much lower,or no cytolytic activity against normal, non-pathogenic cells, may beused for a variety of therapeutic applications with no or little toxicside effects.

One group of cyclic peptides in accordance with the invention arederived from non-α-helical heterogeneous peptides derived fromhomogeneous peptides with an α-helical structure possessing a broadrange cytolytic activity. The present invention thus provides inaccordance with one embodiment, a heterogeneous peptide cyclicderivative comprising both D- and L-amino acid residues having asequence such that a homogeneous open chain peptide comprising only L-or only D-amino acid residues and having the same amino acid sequence assaid heterogeneous peptide, has an α-helix configuration and has a broadspectrum cytolytic activity manifested on a variety of cells; saidhetereogeneous cyclic peptide having a cytolytic activity on only someof the cells on which said homogeneous peptide is cytolytically active.For example, a cytolytic activity of the heterogeneous cyclic peptide ismanifested only on pathogenic cells while having no cytolytic activityon normal cells such as red blood cells.

Examples of non-hemolytic cytolytic cyclic peptides in accordance withthe invention are such which are derived from natural peptides whichhave an α-helical structure and possess a cytolytic activity. Thenon-α-helical cyclic peptides of the invention have a sequenceessentially corresponding to the entire or partial sequence of thenatural peptide in which D-amino acids are incorporated along the N- andC-helices of the molecule and a net positive charge is attained eitherby addition of a positively charged amino acid residue, e.g., lysine,arginine, histidine, for example at the N-terminus and/or of apositively charged group, e.g. aminoalkylamino group such asaminoethylamino, for example at the C-terminus of the molecule, or byneutralization of free carboxyl groups e.g. by converting them to amidegroups. Examples of such natural peptides are melittin and pardaxin, andfragments thereof.

For example, the non-α-helical cyclic peptide may be derived frompardaxin which is a 33-mer peptide or from melittin, which is a 26-merpeptide, the non-α-helical cyclic peptide may be a 33-mer or a 26-merpeptide comprising a sequence corresponding to the entire sequence ofpardaxin or of melittin, respectively, or may be a non-helical cyclicpeptide having a sequence corresponding to a partial sequence ofpardaxin or melittin, e.g., 8–23 mer melittin sequence. In the case of aheterogeneous cyclic peptide derived from pardaxin, the heterogeneouscyclic peptide in accordance with the invention may comprise a partialsequence corresponding to that of pardaxin, comprising as little as 10amino acid residues and ranging between 10 and 24 amino acid residues.

Another group of peptides in accordance with the invention arenon-helical peptides which have a sequence having no natural homologsand are composed of at least one hydrophobic and at least one positivelycharged amino acid and in which sequence at least one amino acid residueis a D-amino acid.

Previous studies with model peptides used to elucidate thestructure-function study of antibacterial peptides focused on threeparameters; helical structure, hydrophobicity and charge (Anzai et al.,1991; Agawa et al., 1991). Each change in one of these parameterssimultaneously resulted in changes in the other two, making it difficultto clarify the unique contribution of each parameter to the overallantibacterial activity. According to the present invention, the effectof the helical structure was eliminated which therefore permitted thestudy of only two parameters, namely, hydrophobicity and net positivecharge, by varying the ratio of leucine and lysine. For this purpose,diastereomers of short model peptides (12 aa. long) containing stretchesof only 1—3 consecutive L-amino acids which are too short to form ana-helical structure, where chosen for investigation.

CD spectroscopy revealed that these Leu/Lys diastereomers are indeedtotally devoid of α-helical structure (data not shown), unlike thediastereomers of melittin and pardaxin of the invention which retain lowα-helical structure. Nevertheless, the Leu/Lys diastereomers exhibitpotent antibacterial activity similar to or greater than that of nativeantibacterial peptides such as dermaseptin S, or the antibiotic drugtetracycline. Moreover, the most potent peptides [D]-L^(3,4,8,10)-K₄L₈and [D]-L^(3,4,8,10)-K₅L₇ (peptides 23 and 24, respectively, of Example3 herein) were devoid of hemolytic activity against the highlycytolytically-susceptible human erythrocytes. It should be noted that[D]-L^(3,4,8,10)-K₃L₉ (peptides 22) is devoid of α-helical structure buthas considerable hemolytic activity which approaches that of the nativecytolytic peptide, pardaxin. This could indicate that the balancebetween hydrophobicity and positive charge compensates for theamphipathic α-helical structure. However, increasing the positive chargedrastically reduced the hemolytic activity while antibacterial activitywas preserved, demonstrating that the amphipathic α-helical structure isnot required for antibacterial activity.

The interaction of the Leu/Lys diastereomers with bothnegatively-charged and zwitterionic phospholipid membranes was examinedin order to elucidate the basis of their selective cytotoxicity againstbacteria. Negatively-charged PE/PG vesicles were used to mimic the lipidcomposition of E. coli (Shaw, 1974), and the zwitterionic PC vesicles tomimic the outer leaflet of human erythrocytes (Verkleij et al., 1973).The biological activity of the Leu/Lys peptides on erythrocytes (FIG.12) and E. coli (Table 5) correlates well with their ability to permeatemodel membranes. The only peptide which permeated PC vesicles was theonly peptide with significant hemolytic activity. These results suggestthat the phospholipid composition of the bacterial membrane plays a rolein permeation by this family of antibacterial peptides. The ability ofantibacterial and non-hemolytic peptides to bind and permeatenegatively-charged but not zwitterionic phospholipid vesicles ischaracteristic of native antibacterial peptides (Gazit et al., 1994),and has been attributed to the fact that the bacterial surface containslipopolysaccharides (LPS, in Gram-negative bacteria), andpolysaccharides (teichoic acids, in Gram-positive bacteria), and theirinner membranes contain phosphatidyl glycerol (PG), all of which arenegatively charged, while normal eukaryotic cells such as erythrocytes,predominantly express the zwitterionic phospholipid PC on their outerleaflet.

The antibacterial peptide magainin is a non-hemolytic peptide, whilemelittin, pardaxin, and a model peptide with a sequence similar to thatof [D]-L^(3,4,8,10)-K₄L₈, but composed of entirely L-amino acids, arehemolytic, mainly due to their high hydrophobicity. When the α-helicalstructure of magainin was disrupted by the introduction of three D-aminoacids, the resulting diastereomer had no antibacterial activity (Chen etal., 1988), even though its net positive charge is similar to that ofnative magainin. Thus, an optimal balance that already exists betweenthe α-helical structure, hydrophobicity and net positive charge ofnative magainin, allows selective antibacterial activity, and any changein one of these properties could cause a loss in magainin'santibacterial activity. Contrastingly, hydrophobicity appears to play amajor role in compensating for the loss of α-helical structure inmelittin, pardaxin and the Leu/Lys diastereomers of the invention.

The results according to the invention suggest a new strategy for thedesign of a repertoire of short, simple, and easily manipulatedantibacterial peptides. Each of the diastereomeric model Leu/Lyspeptides has a unique spectrum of activity (Table 5). The existence of arepertoire of diastereomeric antibacterial peptides will enable one tochoose the most efficacious peptide with regard to the target cell.Furthermore, simultaneous administration of multiple forms ofdiastereomers peptides, acting separately or in concert, also has aselective survival value, and provides a better shielding against awider range of infectious microbes. All the Leu/Lys diastereomersdisplayed increased antibacterial activity against Gram-positive incomparison to Gram-negative bacteria. These results are importantconsidering the increasing resistance of Gram-positive bacteria such asStaphylococcus aureus, enterococci, and pneumococci to conventionalantibiotics (Russell et al., 1995). In addition, unlike the nativeantibacterial peptide dermaseptin S, [D]-L^(3,4,8,10)-K₅L₇ (peptide 24)retained its antibacterial activity in the presence of pooled humanserum.

Diastereomeric peptides should have several advantages over knownantibacterial peptides: (1) The peptides should lack the diversepathological and pharmacological effects induced by α-helical lyticcytolysins. For example, staphylococcus δ-toxin, the antibacterialpeptide alamethicin, cobra direct lytic factor and pardaxin exertseveral histopathological effects on various cells due to pore formationand activation of the arachidonic acid cascade. However, pardaxindiastereomers do not exert these activities. In addition, manyamphipathic α-helical peptides bind to calmodulin and elicit severalcell responses, and even all D-amino acid α-helices, including melittinhave similar activity (Fisher et al., 1994). Diastereomers withdisrupted α-helical structure are not expected to bind to calmodulin;(2) Local D-amino acid substitution would result in controlled clearanceof the antibacterial peptides by proteolytic enzymes, as opposed to thetotal protection acquired by complete D-amino acids substitution (Wadeet al., 1990). Total resistance of a lytic peptide to degradation isdisadvantageous for therapeutic use. Furthermore, the antigenicity ofshort fragments containing D,L amino acids is dramatically altered ascompared to their wholly L or D-amino acid parent molecules (Benkiraneet al., 1993); (3) Total inhibition of bacterial growth induced by thediastereomers, is associated with total lysis of the bacterial wall, asshown by electron microscopy (FIG. 14). Therefore, bacteria might noteasily develop resistance to drugs that trigger such a destructivemechanism; (4) [D]-L^(3,4,8,10)-K₅L₇ (peptide 24) has the ability toperturb the cell wall of bacteria at concentrations lower than theirMIC, as seen by electron microscopy (FIG. 14). The simultaneousadministration of clinically used antibiotics, which have no activitydue to their inability to penetrate the bacterial cell wall, togetherwith peptide 24, may present a solution to this resistance mechanism ofbacteria.

The invention will now be described with reference to some non-limitingdrawings and examples.

EXPERIMENTAL PROCEDURES

(i) Materials. Butyloxycarbonyl-(amino acid)-(phenylacetamido) methylresin was purchased from Applied Biosystems (Foster City, Calif.) andbutyloxycarbonyl (Boc) amino acids were obtained from PeninsulaLaboratories (Belmont, Calif.). Other reagents used for peptidesynthesis included trifluoroacetic acid (TFA, Sigma),N,N-diisopropylethylamine (DIEA, Aldrich, distilled over ninhydrin),dicyclohexylcarbodiimide (DCC, Fluka), 1-hydroxybenzotriazole (HOBT,Pierce) and dimethylformamide (peptide synthesis grade, Biolab). Eggphosphatidylcholine (PC) and phosphatidylserine (PS) from bovine spinalcord (sodium salt-grade I) were purchased from Lipid Products (SouthNutfield, U.K). Egg phosphatidylglycerol (PG) andphosphatidylethanolamine (PE) (Type V, from Escherichia coli) werepurchased from Sigma. Cholesterol (extra pure) was supplied by Merck(Darmstadt, Germany) and recrystallized twice from ethanol.3,3′-Diethylthio-dicarbocyanine iodide [diS-C₂-5] was obtained fromMolecular Probes (Eugene, Oreg.). Native melittin was purchased fromSigma. Commercially available melittin usually contains traces ofphospholipase A₂, which causes rapid hydrolysis of phospholipids.Therefore, special care was taken to remove all the phospholipase A₂from melittin using RP-HPLC. All other reagents were of analyticalgrade. Buffers were prepared in double glass-distilled water.

(ii) Peptide synthesis and purification. Peptides were synthesized by asolid phase method on butyloxycarbonyl-(amino acid)-(phenylacetamido)methyl resin (0.05 meq) (Merrifield et al., 1982). The resin-boundpeptides were cleaved from the resins by hydrogen fluoride (HF), andafter HF evaporation extracted with dry ether. These crude peptidepreparations contained one major peak, as revealed by RP-HPLC, that was50–70% pure peptide by weight. The synthesized peptides were furtherpurified by RP-HPLC ona C₁₈ reverse phase Bio-Rad semi-preparativecolumn (300 Å pore size). The column was eluted in 40 min, using alinear gradient of 10–60% acetonitrile in water, both containing 0.05%TFA (v/v), at a flow rate of 1.8 ml/min. The purified peptides, whichwere shown to be homogeneous (˜95%) by analytical HPLC, were subjectedto amino-acid analysis and to mass spectrometry to confirm theirsequences.

(iii) Transamination of the peptides. The resin-bound peptides as in(ii) above were transaminated with 30% ethylene diamine in DMF for 3days, followed by filtration of the resin, precipitation of theprotected peptides, namely aminoethylamino (TA) peptides, with ether andremoval of the protecting groups with HF. The synthetic TA-peptides werepurified (>95% homogenicity) by reverse-phase HPLC on a C₁₈ column usinga linear gradient of 25–80% acetonitrile in 0.1% TFA, in 40 min, andthen subjected to amino acid analysis to confirm their composition.

(iv) Amidation of the peptides. Resin-bound peptide (20 mg) was treatedfor 3 days with a mixture composed of 1:1 v/v of saturated ammoniasolution (30%) in methanol and DMSO (1:1 v/v) which resulted inamidation of the carboxylate group of the glutamine residue located atthe C-terminus of [D]-V^(5,8),I¹⁷,K²¹-melittin. Thus, peptides wereobtained in which all the protecting groups remained attached, but whoseC-terminal residues were modified by one amide group. The methanol andammonia were evaporated under a stream of nitrogen, and the protectedpeptides were extracted from the resin with DMSO, and precipitated withdry ether. The products were then subjected to HF cleavage and tofurther purification using RP-HPLC as described above.

(v) Preparation of lipid vesicles. Small unilamellar vesicles (SUV) wereprepared by sonication of PC/cholesterol (10:1 w/w) or PC/PS (1:1 w/w)dispersions. Briefly, dry lipid and cholesterol (10:1 w/w) weredissolved in a CHCl₃/MeOH mixture (2:1 v/v). The solvents were thenevaporated under a stream of nitrogen and the lipids (at a concentrationof 7.2 mg/ml) were subjected to a vacuum for 1 h and then resuspended inthe appropriate buffer, by vortexing. The resultant lipid dispersionswere then sonicated for 5–15 min in a bath type sonicator (G1125SP1sonicator, Laboratory Supplies Company Inc., NY) until clear. The lipidconcentrations of the resulting preparations were determined byphosphorus analysis (Bartlett, 1959). Vesicles were visualized using aJEOL JEM 100B electron microscope (Japan Electron Optics Laboratory Co.,Tokyo, Japan) as follows. A drop of vesicles was deposited on acarbon-coated grid and negatively stained with uranyl acetate.Examination of the grids demonstrated that the vesicles were unilamellarwith an average diameter of 20–50 nm (Papahadjopoulos and Miller, 1967).

(vi) Preparation of serum. Blood was collected from five volunteers andallowed to clot at room temperature for 4 h. The blood was thencentrifuged for 15 min at 1500 g, and the serum was removed and pooled.The serum complement was inactivated by heating at 56° C. for 30 min.

(vii) CD Spectroscopy. The CD spectra of the peptides were measured witha Jasco J-500A spectropolarimeter after calibrating the instrument with(+)-10-camphorsulfonic acid. The spectra were scanned at 23° C. in acapped, quartz optical cell with a 0.5 mm path length. Spectra wereobtained at wavelengths of 250 to 190 nm. Eight scans were taken foreach peptide at a scan rate of 20 nm/min. The peptides were scanned atconcentrations of 1.5×10⁻⁵–2.0×10⁻⁵ M in 40% trifluoroethanol (TFE), asolvent that strongly promotes α-helical structure. Fractionalhelicities (Greenfield and Fasman, 1969; Wu et al., 1981) werecalculated as follows:$f_{h} = \frac{\lbrack\theta\rbrack_{222} - \lbrack\theta\rbrack_{222}^{0}}{\lbrack\theta\rbrack_{222}^{100} - \lbrack\theta\rbrack_{222}^{0}}$where [θ]₂₂₂ is the experimentally-observed mean residue ellipticity at222 nm, and the values for [θ]⁰ ₂₂₂ and [θ]¹⁰⁰ ₂₂₂, which correspond to0% and 100% helix content at 222 nm, are estimated to be 2000 and 32000deg·cm²/dmole,

(viii) Antibacterial activity of the peptides. The antibacterialactivity of the diastereomers was examined in sterile 96-well plates(Nunc F96 microtiter plates) in a final volume of 100 μL as follows:Aliquots (50 μl) of a suspension containing bacteria at a concentrationof 10⁶ Colony-Forming Units (CFU)/ml LB (Lauria broth) medium were addedto 50 μL of water or 66% pooled normal human serum in PBS, containingthe peptide in 2-fold serial dilutions. Growth inhibition was determinedby measuring the absorbance at 492 nm with a Microplate autoreader E1309(Bio-tek Instruments), following incubation for 18–20 h at 37° C.Antibacterial activity is expressed as the minimal inhibitoryconcentration (MIC), the concentration at which 100% inhibition ofgrowth was observed after 18–20 h of incubation. The bacteria used were:Escherichia coli D21, Pseudomonas aeruginosa ATCC 27853, Acinetobactercalcoaceticus Ac11, Salmonella typhimurium LT2, Bacillus megateriumBm11, Micrococcus Iuteus ATCC 9341, Bacillus subtilis ATCC 6051.

(ix) Hemolysis of human red blood cells. The peptides were tested fortheir hemolytic activities against human red blood cells (hRBC). FreshhRBC with EDTA were rinsed 3 times with PBS (35 mM phosphate buffer/0.15M NaCl, pH 7.3) by centrifugation for 10 min at 800 g and resuspended inPBS. Peptides dissolved in PBS were then added to 50 μL of a solution ofthe stock hRBC in PBS to reach a final volume of 100 μL (finalerythrocyte concentration, 5% v/v). The resulting suspension wasincubated under agitation for 30 min at 37° C. The samples were thencentrifuged at 800 g for 10 min. Release of hemoglobin was monitored bymeasuring the absorbance of the supernatant at 540 nm. Controls for zerohemolysis (blank) and 100% hemolysis consisted of hRBC suspended in PBSand Triton 1%, respectively.

(x) Visualization of the effects of the peptides on bacteria by electronmicroscopy. Samples containing E. coli (10⁶ CFU/ml) in LB medium wereincubated with the various peptides at their MIC, and one dilution lessthan the MIC, for 16 h, and then centrifuged for 10 min at 3000 g. Thepellets were resuspended and a drop containing the bacteria wasdeposited onto a carbon-coated grid which was then negatively-stainedwith 2% phosphotungstic acid (PTA), pH 6.8. The grids were examinedusing a JEOL JEM 100B electron microscope.

(xi) Membrane permeation induced by the peptides. Membrane permeationwas assessed utilizing the diffusion potential assay (Loew et al., 1983;Sims et al., 1974) as previously described (Shai et al., 1991). In atypical experiment, in a glass tube, 4 μl of a liposomes suspension(final phospholipids concentration of 33 μM), in a K⁺ containing buffer(50 mM K₂SO₄, 25 mM HEPES-SO₄ ⁻², pH 6.8), was diluted in 1 ml of anisotonic K⁺ free buffer (50 mM Na₂SO₄, 25 mM HEPES-SO₄ ⁻², pH 6.8), andthe fluorescent, potential-sensitive dye diS-C₂-5 was then added.Valinomycin (1 μl of 10⁻⁷ M) was added to the suspension in order toslowly create a negative diffusion potential inside the vesicles, whichled to a quenching of the dye's fluorescence. Once the fluorescence hadstabilized, which took 3–10 minutes, peptides were added. The subsequentdissipation of the diffusion potential, as reflected by an increase influorescence, was monitored on a Perkin Elmer LS-50B spectrofluorometer,with the excitation set at 620 nm, the emission at 670 nm, and the gainadjusted to 100%. The percentage of fluorescence recovery, F_(t), wasdefined as:F _(t)=(I _(t) −I ₀ /I _(f) −I ₀)×100where I₀=the initial fluorescence, I_(f)=the total fluorescence observedbefore the addition of valinomycin, and I_(t)=the fluorescence observedafter adding the peptide at time t.

(xii) Binding of peptides to vesicles. The interaction of[D]-V^(5,8),I¹⁷,K²¹-melittin with vesicles consisting of zwitterionic(PC) or negatively charged phospholipids (PC/PS) was characterized bymeasuring changes in the emission intensity of the peptides' intrinsictryptophan in SUV titration experiments. Briefly, SUV were added to afixed amount of peptide (0.5 μM) dissolved in buffer containing 50 mMNa₂SO₄, 25 mM HEPES-SO₄ ⁻², pH 6.8, at 24° C. A 1-cm pathlength quartzcuvette that contained a final reaction volume of 2 ml was used in allexperiments. The fluorescence intensity was measured as a function ofthe lipid/peptide molar ratio (4 separate experiments) on a Perkin-ElmerLS-5 Spectrofluorometer, with excitation set at 280 nm, using a 5 nmslit, and emission set at 340 nm, using a 2.5 nm slit. The bindingisotherms were analysed as a partition equilibrium, using the followingformula:X _(b) =K _(P) C _(f)where X_(b) is defined as the molar ratio of bound peptide (C_(b)) pertotal lipid (C_(L)), K_(P) corresponds to the partition coefficient, andC_(f) represents the equilibrium concentration of the free peptide insolution. For practical purposes, it was assumed that the peptidesinitially were partitioned only over the outer leaflet (60%) of the SUV.Therefore, the partition equation becomes:X _(b) *=K _(P) *C _(f)where X_(b)* is defined as the molar ratio of bound peptide per 60% oftotal lipid and K_(P)* is the estimated surface partition constant. Thecurve resulting from plotting X_(b)* vs. free peptide, C_(f) is referredto as the conventional binding isotherm.

(xiii) Tryptophan quenching experiments. Tryptophan which is sensitiveto its environment has been utilized previously in combination withbrominated phospholipids (Br-PC) to evaluate peptide localization in themembrane (Bolen and Holloway, 1990; De Kroon et al., 1990). Br-PCemployed as quenchers of tryptophan fluorescence are suitable forprobing the membrane insertion of peptides, since they act over a shortdistance and do not drastically perturb the membrane. Melittin and itsdiastereomer, each of which contains one tryptophan residue, were added(final concentration of 0.5 μM) to 2 ml of buffer (50 mM Na₂SO₄, 25 mMHEPES-SO₄ ⁻², pH 6.8) containing 20 μl (50 μM) of Br-PC/PS (1:1 w/w)SUV, thus establishing a lipid/peptide ratio of 100:1. After a 2 minincubation at room temperature, an emission spectrum of the tryptophanwas recorded using a Perkin-Elmer LS-50B Spectrofluorometer, withexcitation set at 280 nm (8 nm slit). SUV composed of PC/PS (1:1 w/w)and which contained 25% of either 6,7 Br-PC, or 9,10 Br-PC, or 10,11Br-PC, were used. Three separate experiments were conducted for eachpeptide. In control experiments, PC/PS (1:1 w/w) SUV without Br-PC wereused.

EXAMPLE 1 Synthesis and Biological Activity of Pardaxin-DerivedDiastereomers

1.1 Synthesis. To examine the role of the α-helical structure of apolycationic cytolysin in its cytotoxicity towards mammalian cells andbacteria, a series of pardaxin-derived peptides were synthesized asdescribed in sections (ii) and (iii) of the Experimental Procedures, andcharacterized for their structure, hemolytic activity on hRBCs,antibacterial activity and effect on the morphology of bacteria.

Pardaxin (par) is a 33-mer peptide of the following sequence:

-   -   Gly-Phe-Phe-Ala-Leu-Ile-Pro-Lys-Ile-Ile-Ser-Ser-Pro-Leu-Phe-Lys-Thr-Leu-Leu-Ser-Ala-Val-Gly-Ser-Ala-Leu-Ser-Ser-Ser-Gly-Gly-Gln-Glu        (SEQ ID NO:98)

Modification of the pardaxin molecule in order to introduce a positivecharge was made by either deleting the acidic C-terminus of pardaxin orconverting the acidic C-terminus of pardaxin or of a fragment thereof toa positive one by reaction of both carboxyl groups of the Glu residue atthe C-terminus with ethylene diamine (TA), and/or adding positivelycharged amino acid residues such as Lys to the N-terminus, in pardaxindiastereomers in which the N-helix and/or the C-helix were altered byeither replacing the residue Pro at position 7 of TApar or of a pardaxinfragment by D-Pro (herein indicated by [D]P⁷), or the two Leu residuesat positions 18 and 19 of TApar or of a pardaxin fragment by D-Leu(herein [D]L¹⁸L¹⁹), or both (herein [D]P⁷L¹⁸L¹⁹). The D-amino acids wereintroduced in the centers of the N- and C-helices.

The following pardaxin-derived diastereomers were found to benon-hemolytic and to exhibit selective cytolytic activity (the bold andunderlined residues are D-amino acids). The peptides will be representedhereinafter by numerals in bold.

-   1. [D]P⁷L¹⁸L¹⁹-TApar of the sequence:    Gly-Phe-Phe-Ala-Leu-Ile-Pro-Lys-Ile-Ile-Ser-Ser-Pro-Leu-Phe-Lys-Thr-Leu-Leu-Ser-Ala-Val-Gly-Ser-Ala-Leu-Ser-Ser-Ser-Gly-Gly-Gln-Glu-(NH—CH₂—CH₂—NH₂)₂    (SEQ ID NO:1)-   2. [D]P⁷L¹⁸L¹⁹[1-22]-TApar of the sequence:    Gly-Phe-Phe-Ala-Leu-Ile-Pro-Lys-Ile-Ile-Ser-Ser-Pro-Leu-Phe-Lys-Thr-Leu-Leu-Ser-Ala-Val-NH—CH₂—CH₂—NH₂    (SEQ ID NO:2)-   3. [D]P⁷L¹⁸L¹⁹[1-22]-par of the sequence:    Gly-Phe-Phe-Ala-Leu-Ile-Pro-Lys-Ile-Ile-Ser-Ser-Pro-Leu-Phe-Lys-Thr-Leu-Leu-Ser-Ala-Val    (SEQ ID NO:3)-   4. K¹[D]P⁷L¹⁸L¹⁹[1-22]-TApar of the sequence:    Lys-Gly-Phe-Phe-Ala-Leu-Ile-Pro-Lys-Ile-Ile-Ser-Ser-Pro-Leu-Phe-Lys-Thr-Leu-Leu-Ser-Ala-Val-NH—CH₂—CH₂—NH₂    (SEQ ID NO:4)-   5. K¹K²[D]P⁷L¹⁸L¹⁹[1-22]-TApar of the sequence:    Lys-Lys-Gly-Phe-Phe-Ala-Leu-Ile-Pro-Lys-Ile-Ile-Ser-Ser-Pro-Leu-Phe-Lys-Thr-Leu-Leu-Ser-Ala-Val-NH—CH₂—CH₂—NH₂    (SEQ ID NO:5)-   6. K¹K²[D]P⁷L¹⁸L¹⁹[1-22]-par of the sequence:    Lys-Lys-Gly-Phe-Phe-Ala-Leu-Ile-Pro-Lys-Ile-Ile-Ser-Ser-Pro-Leu-Phe-Lys-Thr-Leu-Leu-Ser-Ala-Val    (SEQ ID NO:6)-   7. [D]P⁷-[1-11]-TApar of the sequence:    Gly-Phe-Phe-Ala-Leu-Ile-Pro-Lys-Ile-Ile-Ser-NH—CH₂—CH₂—NH₂ (SEQ ID    NO:6)

The following pardaxin derivatives were synthesized and found to behemolytic as shown in Table 1 hereinafter:

-   8. TApar (SEQ ID NO:8) 9. [D]P¹³-TApar (SEQ ID NO:9)-   10. [D]L⁵L¹⁹-TApar (SEQ ID NO:10) 11. [D]P⁷L¹⁹-TApar (SEQ ID NO:11)-   12. [D]P⁷-TApar (SEQ ID NO:12) 13. [D]P⁷-par (SEQ ID NO:13)-   14. [D]L¹⁸L¹⁹-TApar (SEQ ID NO:14) 15. [D]L¹⁸L¹⁹-par (SEQ ID NO:15)-   16. [D]P⁷L¹⁸L¹⁹-par (SEQ ID NO:16)-   17. [D]P⁷[1-22]-TApar (SEQ ID NO:17)

1.2 Determination of the secondary structure of the peptides. Thesecondary structures of the peptides 1, 8, 12, 14, were evaluated fromtheir CD spectra in 40% TFE, a solvent that strongly promotes anα-helical structure, as described in Experimental Procedures, section(vii), and in PBS (35 mM phosphate buffer/0.15 M NaCl, pH 7.0).

The CD spectra of the pardaxin-derived diastereomers are shown in FIG. 1wherein [8] (_), [12] (.......), [14] (-----), and [1] (-.-- -.-.). Asexpected, a dramatic decrease in the α-helix content of the peptides wasobserved as more D-amino acids were incorporated, as reflected by theminima at 208 and 222 nm in 40% TFE. There was a more than 90% decreasein the α-helix content between 8 (TApar) (50% α-helix) and 1([D]P⁷L¹⁸L¹⁹-TApar) (4%). The α-helix contents of 12 ([D]P⁷-TApar) and14 ([D]L¹⁸L¹⁹-TApar) were 25% and 15%, respectively. It should be notedthat proline at position 7 does not introduce a kink in the structurebut rather participates in the formation of the N-helix as revealed byNMR spectroscopy (Zagorski et al., 1991). In PBS, pardaxin gave a lowvalue of ˜12% α-helix content while all the analogues with D-amino acidresidues gave very low signals that could not be attributed to specificstructures (data not shown).

1.3 Hemolytic and antibacterial activity. The pardaxin-derived peptides1–17 were then examined for their hemolytic activity towards the highlysusceptible human erythrocytes, and for their potential to inhibit thegrowth of different species of bacteria, as described in ExperimentalProcedures, sections (ix) and (xviii), respectively. In addition, thecytotoxic bee venom melittin, the antibacterial peptide dermaseptin S,and the antibiotic tetracycline were used as controls.

FIG. 2 shows the dose response curves of the hemolytic activity of thepeptides 1, 8, 12, 14. It is shown that D-amino acids introduced intoTApar dramatically reduced its hemolytic activity, which correlates withthe loss of α-helix content in the corresponding analogues. Peptide 8,TApar, with the highest α-helix content is the most hemolytic, whilePeptide 1, [D]P⁷L¹⁸L¹⁹-TApar, with the lowest α-helix content, ispractically devoid of hemolytic activity up to the maximum concentrationtested (50 μM). The inability to lyse RBCs is characteristic of most ofthe naturally occurring antibacterial peptides such as dermaseptin (seeFIG. 2), magainin and cecropins.

Table 1 gives the MIC (in μM) of the peptides 1–17 for a representativeset of test bacteria, which includes two Gram-negative species,Escherichia coli and Acinetobacter calcoaceticus, and two Gram-positivespecies, Bacillus megaterium and Bacillus subtilis, as well as the %hemolysis at 50 μM peptide. Table 2 gives the MIC (in μM) of thepeptides 1, 8, 12, 14 and of melittin, dermaseptin S and tetracyclinefor some bacterial species The data reveal that despite the dramaticdecrease in the α-helix content and hemolytic activity of thediastereomeric analogues 1–7, they all retained most of the potentantibacterial activity of the parent peptide pardaxin, which iscomparable to that of known native antibacterial peptides.

TABLE 1 Minimal Inhibitory Concentration (μM) and hemolytic activity ofdiastereomers pardaxin analogoues. Minimal Inhibitory Concentration (μM)E. coli A. calcoaceticus B. megaterium M. luteus S. typhimurium P.aeruginosa % hemolysis Peptide (D21) (Ac11) (Bm11) (ATCC 9341) (LT2)(ATCC 27853) at 50 μM peptide 1. 6 6 0.9 12.5 N.D N.D 5 2. 12.5 12.5 2.5  N.D^(a) N.D N.D 0 3. 130 >130 30 N.D >130 >130 0 4. 7.5 7.5 1.5 N.DN.D N.D 0 5. 3.5 3.5 0.75 N.D N.D N.D 0 6. 15 6 6 N.D 120 60 07. >120 >120 30 N.D >120 >120 0 8. 3 3 0.8 5 15 8 100 9. 3 N.D 1.5 N.DN.D N.D 83 10. 3 N.D 1.3 N.D N.D N.D 56 11. 3 N.D 1.5 N.D N.D N.D 82 12.10 5 1.2 5 N.D N.D 49 13. 30 15 3.5 N.D >100 >100 23 14. 3.5 1.5 0.6 2.5N.D N.D 100 15. 15 3.5 1.7 N.D 60 60 44 16. 100 100 50 N.D >100 >100 017. 10 N.D 1 N.D N.D N.D 17 ^(a)N.D, not determined.

TABLE 2 Minimum Inhibitory concentration (μM)^(a) of the peptides.Bacterial species Strain 8 12 14 1 Melittin DermaseptinS TetracyclineEscherichia coli D21 3 10 3.5 6 5 6 1.5 Acinetobacter calcoaceticus Ac113 5 2.5 6 2 3 1.5 Bacillus megaterium Bm11 0.8 1.2 0.6 0.9 0.3 0.5 1.2Bacillus subtilis ATCC-6051 1.5 2 1.5 3 0.6 4 6.5 ^(a)Results are themean of 3 independent experiments each performed in duplicates, withstandard deviation of 20%.

1.4 Membrane destabilization induced by the pardaxin-derived peptides. Acommon property of all of the α-helical, positively charged,naturally-occurring antibacterial peptides studied so far, is theirability to interact and permeate negatively charged phospholipids betterthan zwitterionic phospholipids. The relevance of these findings totheir biological target membranes has been attributed to the fact thatthe surface of bacteria contains lipopolysaccharides (LPS, inGram-negative bacteria), and polysaccharides (teichoic acids, inGram-positive bacteria), both of which are acidic, while normalmammalian cells (e.g., erythrocytes) express the predominantlyzwitterionic phospholipid PC on their outer leaflet. The dissipation ofthe diffusion potential to assess the membrane permeating activity ofthe peptides on both PC and PC/PS phospholipid vesicles (preparedaccording to Experimental Procedures, section v) was assayed asdescribed in Experimental Procedures, section xi. The results shown inFIG. 3 for peptides 1, 8, 12, 14, indicate that D-amino acids introducedinto pardaxin did not significantly affect the ability of the peptidesto permeate phospholipid membranes. However, peptide 1, the onlydiastereomer that is devoid of hemolytic activity but retainsantibacterial activity, permeates negatively charged phospholipidssignificantly better than zwitterionic phospholipids. As such it behavessimilar to native antibacterial peptides, although it is devoid ofα-helical structure. The lack of significant intermediate activitieswith peptides 12 and 14 might be explained by the fact that they bothhave either the hydrophobic N-helix or the amphipathic C-helix intact,which is sufficient to promote strong binding to both types of vesiclesvia hydrophobic interactions.

1.5 Visualization of bacterial lysis using electron microscopy. Theeffect of the pardaxin-derived peptides on the morphology of intact andtreated bacteria was visualized using negative staining electronmicroscopy, as described in Experimental Procedures, section xx. Thepeptides were added to bacteria at or below their MIC concentrationunder the same conditions used in the antibacterial assay (see example1.3 above). Samples were pulled out after an 18 h incubation and wereimmediately fixed and examined by transmission electron microscopy. FIG.4 shows the photographs obtained with the non-hemolytic analogue 1,[D]P⁷L¹⁸L¹⁹-TApar, as an example. It was found that at the MIC peptide 1lysed the bacteria completely, and only small fragments could beobserved (FIG. 4C). However, at concentrations lower than the MIC,patches were observed on the bacterial wall (FIG. 4B). These patchesmight indicate the initial step involved in the lytic process.

EXAMPLE 2 Synthesis and Biological Activity of Melittin-DerivedDiastereomers

2.1 Synthesis. In order to further examine the role of the α-helicalstructure of cytolysins in their cytotoxicity against mammalian cellsand bacteria and to gain insight into the mechanism underlying thiseffect, four diastereomers of melittin (mel) were synthesized.

Melittin is a 26-mer peptide of the sequence:Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-NH₂(SEQ ID NO:99)

Modification of the melittin molecule in order to introduce a positivecharge was made by converting the acidic C-terminus of melittin or of afragment thereof to a positive one by reaction of the carboxyl group atthe C-terminus with ethylene diamine, in melittin diastereomers in whichthe N-helix and the C-helix were altered by replacing the two Valresidues at positions 5 and 8 of melittin, the Ile residue at position17 and the Lys residue at position 21 by D-Val, D-Ile and D-Lys,respectively (herein [D]-V⁵V⁸I¹⁷K²¹).

The following melittin-derived diastereomers were found to benon-hemolytic and to exhibit selective cytolytic activity (the bold andunderlined residues are D-amino acids):

-   18. [D]-V⁵V⁸I¹⁷K²¹-mel of the sequence:    Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-NH₂    (SEQ ID NO:18)-   19. [D]-V⁵V⁸I¹⁷K²¹-mel-COOH of the sequence:    Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-COOH    (SEQ ID NO:19)-   20. [D]-V⁵V⁸I¹⁷K²¹-[1-22]-TAmel of the sequence:    Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-NH—CH₂—CH₂—NH₂    (SEQ ID NO:20)-   21. [D]-V⁵V⁸I¹⁷K²¹-[4-22]-TAmel of the sequence:    Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile--Lys-Arg-NH—CH₂—CH₂—NH₂    (SEQ ID NO:21)

The peptides 18–21 were then characterized with regard to theirstructure, biological function and interaction with bacteria and modelmembranes composed of either zwitterionic or negatively chargedphospholipids.

2.2 CD spectroscopy. The extent of the α-helical structure of thepeptides 18 and 19 was determined from their CD spectra in 40% TFE, asolvent that strongly promotes α-helical structure. As expected, theα-helical content of the diastereomers was much lower (80% decrease)than that of melittin, as reflected by the minima at 208 and 222 nm(FIG. 5). The α-helix content of melittin was 73% compared to 15% and 7%in its diastereomers, 18 and 19, respectively.

2.3 Antibacterial and hemolytic activity of the melittin diastereomers18–21. The hemolytic activity of the peptides 18–21 against hRBC andtheir potential to inhibit the growth of different species of bacteriawere investigated. The antibiotic tetracycline served as a control inthe antibacterial assay. A dose response curve for the hemolyticactivity of the peptides was obtained (FIG. 6). Table 3 gives the MICfor a representative set of test bacteria. It can be seen that theintroduction of D-amino acids into melittin dramatically reduced itshemolytic activity, which paralleled the loss of the α-helical contentin the corresponding analogues. Melittin, with the highest α-helicalcontent was the most hemolytic, while up to the maximum concentrationtested (50 μM), peptides 18 and 19, with the lowest α-helical content,were practically devoid of hemolytic activity. However, despite thedramatic decrease in the hemolytic activity of the melittindiastereomers 18 and 19, they both retained most of the potentantibacterial activity of the parent peptide. Furthermore, theantibacterial activity of peptide 19 was only slightly lower than thatof 18, which indicates that the amide group at the C-terminus ofmelittin does not contribute significantly to the antibacterialactivity. In contrast, it is known that cecropin with a free carboxylicC-terminal has a significant lower antibacterial activity than that ofthe native cecropin with an amidated C-terminal (Li et al., 1988).

TABLE 3 Minimal Inhibitory concentration (μM) and Hemolytic activity ofdiastereomer melittin analogues. Minimal Inhibitory Concentration (μM)E. coli A. calcoaceticus B. megaterium M. luteus B. subtilis % hemolysisPeptide designation (D21) (Ac11) (Bm11) (ATCC 9341) (ATCC 6051) at 50 μMpeptide Melittin 5 20 0.3 0.4 0.4 100 18 12 12 0.8 25 3.5 0 19 18 18 1.250 8 0 20 8 7 0.8 29 N.D 0 21 21 14 1.2 28 N.D 0 Dermaseptin-S 6 3 0.5N.D 4 9 Tetracycline 1.5 1.5 1.2 N.D 6.5 —

2.4 Electron microscopy study of bacterial lysis. The effect of thepeptide 18 on the morphology of intact and treated bacteria wasvisualized using transmission electron microscopy. As shown in FIG. 7,at the MIC, the peptide 18 caused total lysis of the bacteria (FIG. 7C).However, at concentrations lower then the MIC, patches were observed onthe bacterial wall (FIG. 7B). These patches might represent an initialstep in the lytic process.

2.5 Mode of interaction with phospholipid membranes. Since thebiological activities of the peptides 18 and 19 were similar, only themode of interaction of peptide 18 with model phospholipid membranes wascompared to that of melittin, in order to elucidate the basis of themembrane selectively observed. For that purpose the ability of thepeptides to dissipate the diffusion potential created in both PC andPC/PS vesicles were measured, and the partition coefficients of thepeptides with both types of vesicles, and the localization of thepeptide when bound to membranes, were determined.

2.5.1 Membrane permeability induced by the peptides. Variousconcentrations of melittin and peptide 18 were mixed with vesicles thathad been pre-treated with the fluorescent dye, diS-C₂-5, andvalinomycin. The kinetics of the fluorescence recovery was monitoredwith time and the maximum lead reached as a function of peptideconcentration was determined. As shown in FIG. 8, both melittin andpeptide 18 had similar membrane permeating activity with PC/PS vesicles,which demonstrated that introduction of D-amino acids into melittin doesnot affect the ability of the resulting diastereomer to permeatenegatively charged phospholipid (PS/PC) membranes. However, whilemelittin was also highly active with PC vesicles, the diastereomer wastotally devoid of membrane permeating activity with PC vesicles (up tothe maximal concentration tested).

2.5.2 Binding Studies. The inability of the diastereomer 18 to permeatePC vesicles may be due to its inability to bind to PC, or alternatively,it may bind to PC vesicles, but once bound cannot organize intostructures that induce membrane leakage. In order to differentiatebetween the two possibilities, a binding study was conducted. The singleTrp residue at position 19 of peptide 18 was used as an intrinsicfluorescence probe to follow its binding to PC and PC/PS vesicles. Afixed concentration (˜0.5 μM) of the peptide was titrated with thedesired vesicles (PC or PC/PS) and an increase in the fluorescenceintensity was observed if binding occurred. Plotting of the resultingincreases in the fluorescence intensities of Trp as a function oflipid:peptide molar ratios yielded conventional binding curves (FIG.9A). The binding curve of peptide 18 with PC/PS reveals that almost allthe peptide molecules bound to the vesicles at a lipid:peptide molarratio of 100:1. However, with PC vesicles a net increase in thefluorescence of the Trp was not observed even with the maximallipid:peptide molar ratio tested, which indicated that the peptide doesnot bind to PC vesicles. Binding isotherms were constructed by plottingX*_(b) (the molar ratio of bound peptide per 60% of the total lipid)versus C_(f) (the equilibrium concentration of the free peptide in thesolution) (FIG. 5B). The surface partition coefficients were estimatedby extrapolating the initial slopes of the curves to C_(f) values ofzero. The estimated surface partition coefficient, Kp*, of peptide 18was 1.1±0.2×10⁴ M⁻¹ (obtained from 4 measurements). This value issimilar to the value reported for melittin binding tophosphatidylglycerol/phosphatidylcholine (4.5±0.6×10⁴ M⁻¹)(Beschiaschvili and Seelig, 1990).

The shape of the binding isotherm of a peptide can provide informationon the organization of the peptide within membranes (Schwarz et al.,1987). The binding isotherm of peptide 18 bend downward indicating anegative cooperativity. A possible explanation for this negativecooperativity is that a low concentration, peptide 18 binding to PS/PCis enhanced by the negative charge of the phospholipid headgroupscompared to the partition equilibrium with no charge effect. Inaddition, upon binding to the membrane the peptide partially neutralizesthe negative membrane surface charge. However, once the membrane surfacecharge is neutralized, further peptide 18 binding is difficult, sincerepulsion of like charges becomes the dominant factor. Similar resultswere obtained in studies of melittin binding to negatively chargedphospholipid membranes) (Batenburg et al., 1987; Beschiaschvili andSeelig, 1990). Interestingly, unlike melittin which binds strongly alsoto PC vesicles (Kuchinka and Seelig, 1989), peptide 18 did not bind toPC vesicles.

2.6 Tryptophan Quenching Experiments. A tryptophan residue naturallypresent in the sequence of a protein or a peptide can serve as anintrinsic probe for the localization of the peptide within a membrane.Melittin contains a tryptophan residue at position 19, the N-terminalside of the C-helix. With both melittin and peptide 18, the largestquenching of tryptophan fluorescence was observed with 6,7-Br-PC/PSvesicles (FIG. 10). Less quenching was observed with 9,10-Br-PC/PS, andthe least with 11,12-Br-PC/PS. These results indicate that upon bindingto vesicles, the peptides were located near the head groups of thephospholipids.

EXAMPLE 3 Synthesis and Biological Activity of Model Lys/LeuDiastereomers

3.1 Lys/Leu diastereomers design. Six diastereomers of short linearmodel 12-mer peptides composed of varying ratios of lysine and leucinewere synthesized in order (1) to examine whether a balance betweenhydrophobicity and a net positive charge may be a sufficient criterianecessary for selective bacterial lysis, and (2) to gain insight intothe mechanism underlying this effect.

In the first series of model Lys/Leu 12-mer peptides 22–25, one third oftheir sequence was composed of D-amino acid residues. The location ofthe D-amino acids remained constant in all peptides which wasconstructed for maximum disruption of α-helical structure. D-amino acidswere distributed along the peptide, leaving only very short stretches of1–3 consecutive L-amino acids. The following peptides were synthesized:

-   22. [D]-L^(3,4,8,10)-K₃L₉ of the sequence:    Lys-Leu-Leu-Leu-Leu-Leu-Lys-Leu-Leu-Leu-Leu-Lys-NH₂ (SEQ ID NO:22)-   23. [D]-L^(3,4,8,10)-K₄L₈, of the sequence    Lys-Leu-Leu-Leu-Lys-Leu-Leu-Leu-Lys-Leu-Leu-Lys-NH₂ (SEQ ID NO:23)-   24. [D]-L^(3,4,8,10)-K₅L₇, of the sequence    Lys-Leu-Leu-Leu-Lys-Leu-Lys-Leu-Lys-Leu-Leu-Lys-NH₂ (SEQ ID NO:24)-   25. [D]-L^(3,4,8,10)-K₇L₅ of the sequence:    Lys-Lys-Leu-Leu-Lys-Leu-Lys-Leu-Lys-Leu-Lys-Lys-NH₂ (SEQ ID NO:25)

In the second series of model Lys/Leu 12-mer peptides 26–27, two thirdsof their sequence were composed of D-amino acid residues, at the exactpositions of the L-amino acid residues of peptides 23 and 24 as follows:

-   26. [D]-K^(1,5,9,12)L^(2,6,7,11)-K₄L₈, of the sequence:    Lys-Leu-Leu-Leu-Lys-Leu-Leu-Leu-Lys-Leu-Leu-Lys-NH₂ (SEQ ID NO:26)-   27. [D]-K^(1,5,7,9,12)L^(2,6,11)-K₅L₇, of the sequence:    Lys-Leu-Leu-Leu-Lys-Leu-Lys-Leu-Lys-Leu-Leu-Lys-NH₂ (SEQ ID NO:27)

In a third series of model Lys/Leu peptides, a 6-mer and a 8-merdiastereomers were synthesized (peptides 28 and 29, respectively):

-   28. [D]-L^(2,4)-K₂L₄, of the sequence: Lys-Leu-Leu-Leu-Leu-Lys (SEQ    ID NO:28)-   29. [D]-L^(2,4,6)-K₃L₅, of the sequence:    Lys-Leu-Leu-Leu-Lys-Leu-Leu-Lys (SEQ ID NO:29)

Further Lys/Leu diastereomers according to the invention that weresynthesized:

-   30. Lys Leu Leu Leu Lys Leu Lys Leu Lys LeuLeu Lys (SEQ ID NO:30)-   31. Lys Leu Leu LeuLys Leu Lys Leu Lys Leu Leu Lys (SEQ ID NO:31)-   32. Lys Leu Leu Leu Lys Leu Lys Leu Lys LeuLeu Lys (SEQ ID NO:32)

3.2 Synthesis of Lys/Leu diastereomers—The peptides were synthesized asdescribed in Experimental Procedures, section (ii). The peptides werethen characterized with regard to their structure, biological functionand interaction with bacteria and model membranes composed of eitherzwitterionic or negatively charged phospholipids.

3.3 Hydrophobicity. The hydrophobicities and net positive charges of thepeptides 22–25 are listed in Table 4. Mean values of hydrophobicity werecalculated using consensus value of hydrophobicity scale (Eisenberg etal., 1984). As shown in FIG. 11, a direct correlation was found betweenhydrophobicity and the retention time of the peptides, suggesting thatstructure does not significantly contribute to overall hydrophobicinteractions with the stationary phase.

TABLE 4 Hydrophobicity and net charge of the Leu/Lys diastereomers.Peptide Designation Net Charge Hydrophobicity 22. +4 0.12 23. +5 −0.0124. +6 −0.15 25. +8 −0.42

3.4 CD spectroscopy. The extent of the α-helical structure of thediastereomers 22–25peptide was determined from their CD spectra in 40%TFE. As expected, after incorporation of D-amino acids, no signal wasobserved for all the diastereomers, demonstrating the lack of anyspecific secondary structure (data not shown). It is to be noted that ina recent study, a peptide with a sequence identical to that of peptide23, but composed of only L-amino acids, was found to have about 40%α-helical structure in methanol and in DMPC vesicles (Cornut et al.,1994).

3.5 Antibacterial and hemolytic activity of the peptides 22–29. Thehemolytic activity of the peptides 22–29 against hRBC was tested. A doseresponse curve for the hemolytic activity of the peptides 22–25 is shownin FIG. 12 wherein the hemolytic activity of melittin served as acontrol. A direct correlation was found between the hydrophobicity(Table 4) and the hemolytic activity of the diastereomers. Peptide 22,[D]-L^(3,4,8,10)-K₃L₉, which has the highest hydrophobicity, was themost hemolytic peptide. However, its hemolytic activity is very low incomparison to melittin (>60 fold less activity). All the other peptidesshowed no significant hemolytic activity up to the maximum concentrationtested (100 μM). The hemolytic activity of peptides 22–29 is shown inTable 5. It should be noted that although peptide 23,[D]-L^(3,4,8,10)-K₄L₈, is not hemolytic at concentrations >100 fold ofthose required for significant hemolysis by melittin, its entirelyL-amino acid form has been shown in a recent study to have hemolyticactivity similar to that of melittin (˜5 fold less) (Cornut et al.,1994).

The peptides 22–29 were also tested for their antibacterial activityagainst a representative set of bacteria, in which tetracycline,dermaseptin S, and melittin served as controls. The resultant MICs areshown in Table 5. The data show that the antibacterial activity of thediastereomers 22–29 was modulated by the balance between hydrophobicityand positively charged amino acids. Both the most hydrophobic peptide 22and the most hydrophilic peptide 25 displayed the lowest range inantibacterial activity (Table 5). However, peptides 23 and 24 displayedhigh antibacterial activity against most of the bacteria tested with theformer being slightly more potent. Furthermore, each peptide had aunique spectrum of antibacterial activity, and each was active moreagainst Gram-positive as compared to Gram-negative bacteria.

3.6 Synergistic effects between tetracycline and the Lys/Leudiastereomers in serum. To investigate a possible synergisticrelationship between the antibiotic tetracycline and the diastereomers,tetracycline was tested in 2-fold serial dilutions against Pseudomonasaeruginosa (ATCC 27853) in the presence of a constant equimolarconcentration (1 μM) of peptide 24, [D]-L^(3,4,8,10)-K₅L₇. Antibacterialactivity of the mixtures was determined as described in Experimentalprocedures, section (xii).

A synergistic effect was observed between tetracycline and thediastereomer 24. Tetracycline shows little activity against P.aeruginosa. However, when mixed with 1 μM solution of peptide 24, aconcentration which is 10 fold lower than that required for lyticactivity against P. aeruginosa, an eight fold increase in the activityof tetracycline was observed (Table 6). A possible explanation for thesynergistic effect is that the peptide slightly disrupts the bacterialwall which improves partitioning of tetracycline into the bacteria. Thisis supported by electron microscopy studies which show that below itsMIC, peptide 24 causes morphological changes in the bacterial wall (FIG.14). In addition, the effect of pooled human serum on the antibacterialactivity of peptide 24 and the native antibacterial peptide dermaseptinagainst P. aeruginosa and E. coli was found to differ considerably(Table 6). While dermaseptin was 8–10 fold less active in the presenceof serum, peptide 24 retained its antibacterial activity.

TABLE 5 Minimal Inhibitory Concentration (μM) of the peptides. MinimalInhibitory Concentration^(a) (μM) E. Coli A. calcoaceticus P. aeruginosaB. megaterium B. subtilis % Hemolysis Peptide Designation (D21) (Ac11)(ATCC-27853) (Bm11) (ATCC-6051) at 100 μM 22 9 20 125 0.7 1.1 58 23 3.54 10 0.4 0.5 0 24 7 20 10 0.25 2 0 25 80 200 >200 1 100 0 26 4 N.D N.D0.5 N.D 0 27 7 N.D N.D 0.2 N.D 0 28 200 N.D N.D 50 N.D 0 29 3 N.D N.D 3N.D 0 Dermaseptin S 6 3 25 0.5 4 — Melittin 5 2 25 0.3 0.6 —Tetracycline 1.5 1.5 50 1.2 6.5 — ^(a)Results are the mean of 3independent experiments each performed in duplicates, with standarddeviation of 20%

TABLE 6 Minimal Inhibitory Concentration (μM)^(a) in the presence ofhuman serum and synergistic activity of peptide 24 Minimal InhibitoryConcentration (μM) P. aeruginosa (ATCC-27853) E. coli (D21) PeptideDesignation 0% Serum 33% Serum 0% Serum 33% Serum 24 10 10 7 7Dermaseptin S 25 200 6 50 Tc^(b) 50 Tc + 24 (1 μM) 6 ^(a)Results are themean of 2 independent experiments each performed in duplicates, withstandard deviation of 20%. ^(b)Tc - Tetracycline

3.7 Peptide-induced membrane permeation. Various concentrations ofpeptides were mixed with vesicles that had been pretreated with thefluorescent dye, diS-C₂-5, and valinomycin. The kinetics of thefluorescence recovery was monitored and the maximum fluorescence levelwas determined as a function of peptide concentration (FIG. 13).PC/cholesterol vesicles (10:1) served as a model of the phospholipidcomposition of the outer erythrocyte leaflet (Verkleij et al., 1973),and PE/PG vesicles (7:3) was used to mimic the phospholipid compositionof E. coli (Shaw, 1974). A direct correlation was found between thepotential of the peptides to permeate model phospholipid membranes andtheir lytic activity against erythrocytes and E. coli. Only thehemolytic peptide 22 permeated the zwitterionic phospholipid vesicles.Furthermore, the ability of the peptides to permeate PE/PG vesiclescorrelates with the antibacterial activity of the peptides against E.coli (Table 5). Peptide 24, which has the lowest antibacterial activity,also had significantly decreased ability to permeate PE/PG vesiclescompared to the other three peptides 22–24.

3.8 Electron microscopy study of bacterial lysis. The effect of thediastereomers 22–25 on the morphology of treated E. coli was visualizedusing transmission electron microscopy. All the peptides caused totallysis of the bacteria at the MIC (data not shown). However, when thepeptides were utilized at concentrations corresponding to 80% of theirMIC, some differences in the morphology of the treated bacteria wereobserved, depending upon the peptide used. The most hydrophobic peptide22 caused the most damage to the cell wall and membranes, while theleast hydrophobic peptide 25 only caused local perturbations (FIG. 14).

EXAMPLE 4 Synthesis and Biological Activity of Model Lys/Ala and Lys/ValDiastereomers

4.1 Diastereomer design. To further examine whether modulatinghydrophobicity and the net positive charge of linear cytotoxic peptidesis sufficient to confer selective antibacterial activity, two furthermodel 12-mer peptides 33 and 34–37, composed of Lys/Ala or Lys/Valresidues, respectively, with at least one third of their sequences beingof D-Ala or D-Val residues, were synthesized:

-   33. [D]-A^(3,4,8,10)-K₄A₈ of the sequence:    Lys-Ala-Ala-Ala-Lys-Ala-Ala-Ala-Lys-Ala-Ala-Lys-NH₂ (SEQ ID NO:33)-   34. [D]-V^(3,4,8,10)-K₄V₈ of the sequence:    Lys-Val-Val-Val-Lys-Val-Val-Val-Lys-Val-Val-Lys-NH₂ (SEQ ID NO:34)-   35. Lys Val Val Val Lys Val Lys ValLys Val Val Lys (SEQ ID NO:35)-   36. Lys Val Val Val Lys Val Lys Val Lys Val Val Lys (SEQ ID NO:36)-   37. Lys Val Val Val Lys Val Lys Val Lys Val Val Lys (SEQ ID NO: 37)

4.2 Synthesis. The Lys/Ala and Lys/Val diastereomers were synthesized asdescribed in Experimental Procedures, section (ii).

4.3 Antibacterial and hemolytic activity. Peptides 33 and 34 were testedagainst E. coli and B. megaterium and hRBC. The results in Table 7 showthat both model diastereomers are antibacterial and non-hemolytic:

TABLE 7 Minimal Inhibitory Concentration (μM) and hemolytic activity ofthe peptides 28 and 29 Minimal Inhibitory Concentration (μM) E. coli B.megaterium % hemolysis Peptide Designation (D21) (Bm11) at 100 μM 33 121 0 34 3.5 0.8 0

EXAMPLE 5 Synthesis of Further Model Diastereomers

The following model diastereomers according to the invention composed ofsequences of 6, 8, 12, 14, 16, 19, 25, 26 and 30 residues of two, threeor more different amino acids, were synthesized:

-   38. Lys Leu Ile Leu Lys Leu (SEQ ID NO: 38)-   39. Lys Val Leu His Leu Leu (SEQ ID NO:39)-   40. Leu Lys Leu Arg Leu Leu (SEQ ID NO: 40)-   41. Lys Pro Leu His Leu Leu (SEQ ID NO:41)-   42. Lys Leu Ile Leu Lys Leu Val Arg (SEQ ID NO: 42)-   43. Lys Val Phe His Leu Leu His Leu (SEQ ID NO: 43)-   44. His Lys Phe Arg Ile Leu Lys Leu (SEQ ID NO: 44)-   45. Lys Pro Phe His Ile Leu His Leu (SEQ ID NO:45)-   46. Lys Ile Ile Ile Lys Ile Lys Ile Lys Ile Ile Lys (SEQ ID NO:46)-   47. Lys Ile Ile Ile Lys Ile Lys Ile Lys Ile Ile Lys (SEQ ID NO: 47)-   48. Lys Ile Ile Ile Lys Ile Lys Ile Lys Ile Ile Lys (SEQ ID NO: 48)-   49. Lys Ile Pro Ile Lys Ile Lys Ile Lys Ile Pro Lys (SEQ ID NO: 49)-   50. Lys Ile Pro Ile Lys Ile Lys Ile Lys Ile Val Lys (SEQ ID NO:50)-   51. Arg Ile Ile Ile Arg Ile Arg Ile Arg Ile Ile Arg (SEQ ID NO:51)-   52. Arg Ile Ile Ile Arg Ile Arg Ile Arg Ile Ile Arg (SEQ ID NO:52)-   53. Arg Ile Ile Ile Arg Ile Arg Ile Arg Ile Ile Arg (SEQ ID NO:53)-   54. Arg Ile Val Ile Arg Ile Arg Ile Arg Leu Ile Arg (SEQ ID NO:54)-   55. Arg Ile Ile Val Arg Ile Arg Leu Arg Ile Ile Arg (SEQ ID NO:55)-   56. Arg Ile Gly Ile Arg Leu Arg Val Arg Ile Ile Arg (SEQ ID NO:56)-   57. Lys Ile Val Ile Arg Ile Arg Ile Arg Leu Ile Arg (SEQ ID NO:57)-   58. Arg Ile Ala Val Lys Trp Arg Leu Arg Phe Ile Lys (SEQ ID NO:58)-   59. Lys Ile Gly Trp Lys Leu Arg Val Arg Ile Ile Arg (SEQ ID NO:59)-   60. Lys Lys Ile Gly Trp Leu Ile Ile Arg Val Arg Arg (SEQ ID NO:60)-   61. Arg Ile Val Ile Arg Ile Arg Ile Arg Leu Ile Arg Ile Arg (SEQ ID    NO:61)-   62. Arg Ile Ile Val Arg Ile Arg Leu Arg Ile Ile Arg Val Arg (SEQ ID    NO:62)-   63. Arg Ile Gly Ile Arg Leu Arg Val Arg Ile Ile Arg Arg Val (SEQ ID    NO:63)-   64. Lys Ile Val Ile Arg Ile Arg Ala Arg Leu Ile Arg IIe Arg Ile Arg    (SEQ ID NO:64)-   65. Arg Ile Ile Val Lys Ile Arg Leu Arg Ile Ile Lys Lys Ile Arg Leu    (SEQ ID NO:65)-   66. Lys Ile Gly Ile Lys Ala Arg Val Arg Ile Ile Arg Val Lys Ile Ile    (SEQ ID NO:66)-   67. Arg Ile Ile Val His Ile Arg Leu Arg Ile Ile His His Ile Arg Leu    (SEQ ID NO:67)-   68. His Ile Gly Ile Lys Ala His Val Arg Ile Ile Arg Val His Ile Ile    (SEQ ID NO:68)-   69. Arg Ile Tyr Val Lys Ile His Leu Arg Tyr Ile Lys Lys Ile Arg Leu    (SEQ ID NO:69)-   70. Lys Ile Gly His Lys Ala Arg Val His Ile Ile Arg Tyr Lys Ile Ile    (SEQ ID NO:70)-   71. Arg Ile Tyr Val Lys Pro His Pro Arg Tyr Ile Lys Lys Ile Arg Leu    (SEQ ID NO: 71)-   72. Lys Pro Gly His Lys Ala Arg Pro His Ile Ile Arg Tyr Lys Ile Ile    (SEQ ID NO: 72)-   73. Lys Ile Val Ile Arg Ile Arg Ile Arg Leu Ile Arg Ile Arg Ile Arg    Lys Ile Val (SEQ ID NO: 73)-   74. Arg Ile Ile Val Lys Ile Arg Leu Arg Ile Ile Lys Lys Ile Arg Leu    Ile Lys Lys (SEQ ID NO: 74)-   75. Lys Ile Gly Trp Lys Leu Arg Val Arg Ile Ile Arg Val Lys Ile Gly    Arg Leu Arg (SEQ ID NO: 75)-   76. Lys Ile Val Ile Arg Ile Arg Ile Arg Leu Ile Arg Ile Arg Ile Arg    Lys Ile Val Lys Val Lys Arg Ile Arg (SEQ ID NO:76)-   77. Arg Phe Ala Val Lys Ile Arg Leu Arg Ile Ile Lys Lys Ile Arg Leu    Ile Lys Lys Ile Arg Lys Arg Val Ile Lys (SEQ ID NO:77)-   78. Lys Ala Gly Trp Lys Leu Arg Val Arg Ile Ile Arg Val Lys Ile Gly    Arg Leu Arg Lys Ile Gly Trp Lys Lys Arg Val Arg Ile Lys (SEQ ID NO:    78)-   79. Arg Ile Tyr Val Lys Pro His Pro Arg Tyr Ile Lys Lys Ile Arg Leu    (SEQ ID NO: 79)-   80. Lys Pro Gly His Lys Ala Arg Pro His Ile Ile Arg Tyr Lys Ile Ile    (SEQ ID NO: 80)-   81. Lys Ile Val Ile Arg Ile Arg Ile Arg Leu Ile Arg Ile Arg Ile Arg    Lys Ile Val (SEQ ID NO:81)-   82. Arg Ile Ile Val Lys Ile Arg Leu Arg Ile Ile Lys Lys Ile Arg Leu    Ile Lys Lys (SEQ ID NO: 82)-   83. Arg Ile Tyr Val Ser Lys Ile Ser Ile Tyr Ile Lys Lys Ile Arg Leu    (SEQ ID NO: 83)-   84. Lys Ile Val Ile Phe Thr Arg Ile Arg Leu Thr Ser Ile Arg Ile Arg    Ser Ile Val (SEQ ID NO: 84)-   85. Lys Pro Ile His Lys Ala Arg Pro Thr Ile Ile Arg Tyr Lys Met Ile    (SEQ ID NO: 85)

EXAMPLE 6 Synthesis and Biological Activity of Cyclic Diastereomers

6.1 Design. The following cyclic derivatives of diastereomers ofpardaxin fragments with cysteine residues at both the N- and C-terminiwere synthesized:

-   86. Cyclic K¹[D]P⁷L¹⁸L¹⁹[1-22]-par of the sequence:    Cys-Lys-Gly-Phe-Phe-Ala-Leu-Ile-Pro-Lys-Ile-Ile-Ser-Ser-Pro-Leu-Phe-Lys-Thr-Leu-Leu-Ser-Ala-Val-Cys    (SEQ ID NO: 86)-   87. Cyclic K¹K²[D]P⁷L¹⁸L¹⁹[1-22]-par of the sequence:    Cys-Lys-Lys-Gly-Phe-Phe-Ala-Leu-Ile-Pro-Lys-Ile-Ile-Ser-Ser-Pro-Leu-Phe-Lys-Thr-Leu-Leu-Ser-Ala-Val-Cys    (SEQ ID NO: 87)-   88. Cyclic K¹K²K³[D]P⁷L¹⁸L¹⁹[1-22]-par of the sequence:    Cys-Lys-Lys-Lys-Gly-Phe-Phe-Ala-Leu-Ile-Pro-Lys-Ile-Ile-Ser-Ser-Pro-Leu-Phe-Lys-Thr-Leu-Leu-Ser-Ala-Val-Cys    (SEQ ID NO: 88)

The following cyclic derivatives of diastereomers of different aminoacid residues with cysteine residues at both the N- and C-termini weresynthesized:

-   89. Cyclic Cys Arg Ile Val Ile Arg Ile Arg Ile Arg Leu Ile Arg Ile    Arg Cys (SEQ ID NO: 89)-   90. Cyclic Cys Lys Pro Gly His Lys Ala Arg Pro His Ile Ile Arg Tyr    Lys Ile Ile Cys (SEQ ID NO:90)-   91. Cyclic Cys Arg Phe Ala Val Lys Ile Arg Leu Arg Ile Ile Lys Lys    Ile Arg Leu Ile Lys Lys Ile Arg Lys Arg Val Ile Lys Cys (SEQ ID NO:    91)-   92. Cyclic Cys Lys Leu Leu Leu Lys Leu Leu Leu Lys Leu Leu Lys Cys    (SEQ ID NO:92)-   93. Cyclic Cys Lys Leu Leu Leu Lys Leu Lys Leu Lys Leu Leu Lys Cys    (SEQ ID NO:93)

The following cyclic derivatives of diastereomers of different aminoacid residues without cysteine residues at both the N- and C-terminiwere synthesized:

-   94. HN-Lys Leu Leu Leu Lys Leu Leu Leu Lys Leu Leu Lys-CO (SEQ ID    NO:94)-   95. HN-Lys Leu Leu Leu Lys Leu Lys Leu Lys Leu Leu Lys-CO (SEQ ID    NO:95)

6.2 Synthesis of the cyclic diastereomers. The cyclic peptides weresynthesized by a solid-phase method as described in ExperimentalProcedures, section (ii), without or with cysteine residues at both theN and C-termini of the peptides. The cyclization without cystein wascarried out by protecting the N-terminal, activating the C-terminal,deprotection of the N-terminal and reaction of the C- and N-terminalgroups while still bound to the resin. After HF cleavage and RP-HPLCpurification the peptides were solubilized at low concentration in PBS(pH 7.3), and cyclization was completed after 12 h. The cyclic peptideswere further purified on RP-HPLC and subjected to amino acid analysis toconfirm their composition, and SDS-PAGE to confirm their monomericstate.

6.3 Antibacterial and hemolytic activity. Peptides 86–88 were testedagainst E. coli and B. megaterium and hRBC. The results in Table 8 showthat all three cyclic pardaxin-derived diastereomers are antibacterialand non-hemolytic:

TABLE 8 Minimal Inhibitory Concentration (μM) and hemolytic activity ofthe cyclic pardaxin-derived diastereomers. Minimal InhibitoryConcentration (μM) E. coli B. megaterium % hemolysis Peptide Designation(D21) (Bm11) at 100 μM 86 30 10 0 87 15 6 0 88 7.5 2 0

6.4 Antibacterial and hemolytic activity. Peptides 92–95 were testedagainst E. coli, B. subtilis and P aeruginosa. The results in Table 8ashow that all four cyclic diastereomers are antibacterial andnon-hemolytic:

TABLE 8a Minimal Inhibitory Concentration (μM) and hemolytic activity ofthe cyclic diastereomers. Minimal Inhibitory Concentration (μM) %hemolysis Peptide Designation E. coli B. subtilis P. aeruginosa at 50 μM92 12.5 1.2 25 0 93 15 5 25 0 94 12.5 1.5 30 0 95 15 6 20 0

EXAMPLE 7 Synthesis and Biological Activity of Bundled Lys/Leu PeptideDiastereomers.

7.1 Design. Using as template peptide 23 and as monomers peptide 23 or24 with an additional cysteine residue at the C-terminus (23C and 24C,respectively, the following bundle-sequences were produced:

-   96. ([D]-L^(3,4,8,10)-K₄L₈C)₅ [D]-L^(3,4,8,10)-K₄L₈ of the sequence:    (Lys-Leu-Leu-Leu-Lys-Leu-Leu-Leu- Lys-Leu-Leu-Lys-Cys-NH₂)₅    Lys-Leu-Leu-Leu-Lys-Leu-Leu-Leu-Lys-Leu-Leu-Lys-NH₂ (SEQ ID    NOS:96and 23)-   97. ([D]-L^(3,4,8,10)-K₅L₇C)₅ [D]-L^(3,4,8,10)-K₄L₉ of the sequence:    (Lys-Leu-Leu-Leu-Lys-Leu-Lys-Leu- Lys-Leu-Leu-Lys-Cys-NH₂)₅    Lys-Leu-Leu-Leu-Lys-Leu-Leu-Leu-Lys-Leu-Leu-Lys-NH₂ (SEQ ID    NOS:97and 24)

7.2 Synthesis. In order to produce template-bound diastereomers, 1:1molar ratio of DCC and bromoacetic acid were allowed to react in DMSO at25° C. for 1 h. The template (peptide 23) was added to the reactionmixture and left under agitation for 12 h after which the DMSO waslyophilized. The remaining bromoacetic acid was extracted with dryether. The template was then reacted with excess of diastereomers 23Cand 24C with cysteine residue at their C-terminus, in PBS pH 7.3 at 25°C. for 1 h. The template-bound diastereomers 96 and 97 were furtherpurified on RP-HPLC, and examined on SDS-PAGE to confirm theiraggregation state.

7.3 Antibacterial and hemolytic activity. The template-bounddiastereomers diastereomers 96 and 97 were tested against E. coli and B.megaterium and hRBC. The results in Table 9 show that both bundlesequences are antibacterial and non-hemolytic.

TABLE 9 Minimal Inhibitory Concentration (μM) and hemolytic activity ofthe bundles. Minimal Inhibitory Concentration (μM) E. coli B. megaterium% hemolysis Peptide Designation (D21) (Bm11) at 100 μM 96 0.2 0.05 0 970.1 0.02 0

EXAMPLE 8 Synthesis and Biological Activity of Mixtures of Lys/Leu12-mer Peptide Diastereomers

Peptides are synthesized by a solid phase method as described inExperimental Procedures, section (ii) above. At each coupling step amixture composed of 1 eq each of lysine, leucine and D-leucine was addedto the reaction. The synthesis resulted in a mixture of 3¹² differentpeptides. After HF cleavage the peptides were extracted with doubledistilled water (ddw) and lyophilized.

The mixture of the Lys/Leu 12-mer peptide diastereomers was testedagainst E. coli D21 (MIC: 15 μg/ml) and B. megaterium Bm11 D21 (MIC: 3μg/ml) and hRBC (0% hemolysis at 100 μM). As expected, the mixture hadantibacterial activity but was non-hemolytic.

EXAMPLE 9 Synthesis and Biological Activity of Lys/Leu/D-Leu RandomCopolymers

In order to produce diastereomers of polymers of different sizes, excessof N-carboxyanhydride residues over initiator free amino acids wereallowed to polymerize in DMF at 25° C. for 4 h (Katchalski and Sela,1958). Polymers consisting of different ratios of lysine, leucine andD-leucine were produced using different ratios oflysine-N-carboxyanhydride, leucine-N-carboxyanhydride andD-leucine-N-carboxy anhydride. Three of such polymers and theirantibacterial and hemolytic activity are shown in Table 10.

TABLE 10 Minimal Inhibitory Concentration (μM) and hemolytic activity ofthe Lys/Leu/D-Leu copolymers. Minimal Inhibitory Concentration (μg/ml)Amino Acids Ratio (Molar) E. coli megaterium % hemolysis Lys:Leu:[D]-Leu(D21) (Bm11) at 100 μM 1:1:1 90 15 0 2:1:1 35 8 0 3:1:1 80 20 0

EXAMPLE 10 Antifungal Activity of the Diastereomers

The antifungal activity of the pardaxin-derived peptides 1 and 16 (seeExample 1 above) was examined in sterile 96-well plates (Nunc F96microtiter plates) in a final volume of 100 μL as follows: Fiftymicroliters of a suspension containing fungi at concentration of 1×10⁶Colony-Forming Units (CFU)/ml in culture medium (Sabouraud's glucosebroth medium) was added to 50 μL of water containing the peptide inserial 2-fold dilutions in water. Inhibition of growth was determined bymeasuring the absorbance at 492 nm with a Microplate autoreader E1309(Bio-tek Instruments), after an incubation time of 48 h at 30° C.Antifungal activities were expressed as the minimal inhibitoryconcentration (MIC), the concentration at which 100% inhibition ofgrowth was observed after 48 h of incubation. The fungi used were:Candida albicans (IP886-65) and Cryptococcus neoformans (IP960-67). Asshown in Table 11, both peptides 1 and 16 showed antifungal activity.

TABLE 11 Minimal Inhibitory Concentration (μM) of the diastereomers 1and 16 against fungi. Minimal Inhibitory Concentration (μM) Candidaalbicans Cryptococcusneoformans Peptide Designation (IP886-65)(IP960-67) 1 35 50 16 120 150

EXAMPLE 11 Anticancer Activity of the Diastereomers

The anticancer activity of the Lys/Leu diastereomers 23 and 24 (seeExample 3 above) was examined against mouse adenocarcinomas. Cells wereseeded at 5–10 000/well in 96-well microtiter plates in Dulbecco'smodified Eagle's medium. After the cells had attached, 20 μl of dilutedpeptide solution in normal saline were transfected to the well to givefinal concentrations ranging from 20 to 150 μM. Following 1 h incubationwith the peptides, the viability of the cancer cell was measured byTrypan blue (0.1% w/v) vital staining assay. In control experiments thepeptide solvent alone was added to the cells. Anticancer activities wereexpressed as the minimal inhibitory concentration (MIC), theconcentration at which 100% inhibition of growth was observed after 1 hof incubation. The results in Table 12 show that both peptides areactive against malignant cells.

TABLE 12 Minimal Inhibitory Concentration (μM) of the diastereomersagainst mouse adenocarcinoma. Minimal Inhibitory Concentration (μM)Peptide Designation mouse adenocarcinoma 23 50 24 80

EXAMPLE 12 Activity of the Diastereomers Against Leishmania mexicana

The melittin-derived diastereomer peptide 20 (see Example 2 above) andthe Leu/Lys diastereomer peptide 23 (see Example 3 above) were testedagainst Leishmania. Promastigotes of the Leishmania mexicana NR strainto be assayed were cultured at 27° C. in RPMI 1640 medium supplementedwith 10% fetal bovine serum. Parasite were harvested by centrifugationat 1200×g for 10 min at 4° C. and washed twice with PBS (50 mM sodiumphosphate, 150 mM NaCl, pH 7). The washed promastigotes were counted ina hemocytometer and adjusted to 1×10⁶ parasites/ml. Aliquotes of thissuspension were assayed in a final volume of 100 μl by counting living(motile) cells after 24 h of incubation at 26° C. in the absence orpresence of various concentrations of the diastereomers. Anti-Leishmaniaactivities were expressed as the minimal inhibitory concentration (MIC),the concentration at which 100% death was observed after 24 h ofincubation. It was found that for peptide 23 the MIC is 17 μM and forpeptide 20 the MIC is 32 μM.

EXAMPLE 13 Antiviral Activity of the Diastereomer 23

Sendai virus (Z strain) was grown in the allantoic sac of 10–11 day oldembryonated chicken eggs, harvested 48 h after injection and purified.The virus was resuspended in buffer composed of 160 mM NaCl, 20 mMtricine, pH 7.4, and stored at −70° C. Virus haemagglutinating activitywas measured in haemagglutinating units (HAU). One microliter contained˜60000 HAU. Fresh human blood was obtained from a blood bank and storedfor up to 1 month at 4° C. Prior to use, erythrocytes were washed twicewith PBS pH 7.2, and diluted to the desired concentration (% v/v) withthe same buffer. Virions, erythrocytes and peptides were mixed indifferent orders of addition and various amounts. The final incubationwas always at 37° C. for 60 min. followed by centrifugation at 5700 gfor 10 min to remove intact cells. In all cases duplicate samples wereused and two aliquots were taken from the supernatant of each sample totwo wells of a 96-well plate. The amount of hemoglobin release wasmonitored by measuring the absorbance of the wells with the ELISA, platereader at 540 nm. Antiviral activity was expressed as the minimalinhibitory concentration (MIC), the concentration at which no release ofhemoglobin was observed after incubation. It was found that for theLys/Leu diastereomer peptide 23 the MIC is 80 μM.

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1. A peptide selected from the group consisting of: (A) a non-naturalsynthetic peptide having from 6 to 12 amino acid residues or anon-natural synthetic cyclic peptide having from 6 to 14 amino acidresidues and a net positive charge which is greater than +1, saidpeptide consisting of hydrophobic amino acid residues excepting glycineand tyrosine, and positively charged amino acid residues, wherein atleast one but not all of such amino acid residues is a D-amino acid,said peptide having a ratio of hydrophobic to positively charged aminoacids such that the peptide is cytolytic to pathogenic cells but doesnot cause cytolysis of red blood cells, and having a sequence of aminoacids such that the same amino acid sequence in which each residue is inthe L-configuration is not found in nature, and cyclic derivativesthereof having from 6 to 14 amino acid residues, with the proviso thatsaid peptide is not that of SEQ ID NO:1, SEQ ID NO:12, SEQ ID NO:14, orSEQ ID NO:23; (B) a non-natural synthetic peptide having from 6 to 12amino acid residues or a non-natural synthetic cyclic peptide havingfrom 6 to 14 amino acid residues and a net positive charge which isgreater than +1, said peptide consisting of hydrophobic amino acidresidues excepting glycine and tyrosine, positively charged amino acidresidues, and polar amino acid residues, wherein at least one but notall of such amino acid residues is a D-amino acid, said peptide having aratio of hydrophobic to positively charged amino acids such that thepeptide is cytolytic to pathogenic cells but does not cause cytolysis ofred blood cells, and having a sequence of amino acids such that the sameamino acid sequence in which each residue is in the L-configuration isnot found in nature, and cyclic derivatives thereof having from 6 to 14amino acid residues, with the proviso that said peptide is not that ofSEQ ID NO:1, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:23; (C) a randomcopolymer having a net positive charge which is greater than +1, saidrandom copolymer consisting of a hydrophobic L-amino acid, a positivelycharged L-amino acid and a D-amino acid in a ratio of hydrophobic topositively charged amino acids such that the copolymer is cytolytic topathogenic cells but does not cause cytolysis of red blood cells; and(D) a mixture of a plurality of peptide diastereomers, each peptidehaving at least 6 amino acids and having a net positive charge which isgreater than +1, said peptide comprising a hydrophobic L-amino acid, apositively charged L-amino acid and a D-amino acid, said mixture beingobtained by solid phase synthesis wherein at each coupling step amixture composed of 1 eq of each of the amino acids is added to thereaction, followed by HF cleavage.
 2. A peptide according to claim 1consisting of a non-natural synthetic peptide having from 6 to 12 aminoacid residues or a non-natural synthetic cyclic peptide having from 6 to14 amino acid residues and a net positive charge which is greater than+1, said peptide consisting of hydrophobic amino acid residues exceptingglycine and tyrosine, and positively charged amino acid residues whereinat least one but not all of such amino acid residues is a D-amino acid,said peptide having a ratio of hydrophobic to positively charged aminoacids such that the peptide is cytolytic to pathogenic cells but doesnot cause cytolysis of red blood cells, and having a sequence of aminoacids such that the same amino acid sequence in which each residue is inthe L-configuration is not found in nature, and cyclic derivativesthereof having from 6 to 14 amino acid residues, with the proviso thatsaid peptide is not that of SEQ ID NO:1, SEQ ID NO:12, SEQ ID NO:14, orSEQ ID NO:23.
 3. The peptide according to claim 2, wherein thepositively charged amino acid residues are selected from the groupconsisting of lysine, arginine and histidine, and the hydrophobic aminoacid residues are selected from the group consisting of leucine,isoleucine, alanine, valine, phenylalanine, proline, and tryptophan. 4.The peptide according to claim 3, in which each of the hydrophobic aminoacid residues is leucine or valine, and each of the positively chargedamino acid residues is lysine.
 5. The peptide according to claim 4,being a diastereomer of a 12-mer peptide in which the hydrophobic aminoacid is valine and the positively charged amino acid is lysine, in whichat least one third of the sequence is composed of D-amino acids, or acyclic derivative thereof.
 6. The peptide according to claim 5consisting of a Val/Lys diastereomer selected from the group of peptidesconsisting of the amino acid sequences of SEQ ID NO:34, SEQ ID NO:35,SEQ ID NO:36, and SEQ ID NO:37.
 7. The peptide according to claim 4,being a diastereomer of a 6-mer, 8-mer or 12-mer peptide in which thehydrophobic amino acid is leucine and the positively charged amino acidis lysine, in which at least one third of the sequence, but not the fullsequence, is composed of D-amino acids, or a cyclic derivative thereof,but expecting the peptide herein designated 23:Lys-Leu-Leu-Leu-Lys-Leu-Leu-Leu-Lys-Leu-Leu-Lys-NH₂ (SEQ ID NO:23). 8.The peptide according to claim 7 consisting of a Leu/Lys diastereomerselected from the group of peptides consisting of those hereindesignated 24 to 29 (SEQ ID NO:24–29), of the sequences: 24)Lys-Leu-Leu-Leu-Lys-Leu-Lys-Leu-Lys-Leu-Leu-Lys-NH₂, 25)Lys-Lys-Leu-Leu-Lys-Leu-Lys-Leu-Lys-Leu-Lys-Lys-NH₂, 26)Lys-Leu-Leu-Leu-Lys-Leu-Leu-Leu-Lys-Leu-Leu-Lys-NH₂, 27)Lys-Leu-Leu-Leu-Lys-Leu-Lys-Leu-Lys-Leu-Leu-Lys-NH₂, 28)Lys-Leu-Leu-Leu-Leu-Lys, and 29) Lys-Leu-Leu-Leu-Lys-Leu-Leu-Lys.
 9. Thepeptide according to claim 7 selected from the group consisting of a6-mer diastereomer in which the ratio of leucine to lysine is 64%:36%and a 12-mer diastereomer in which the ratio of leucine to lysine is66%:34%.
 10. The peptide according to claim 7 consisting of a cyclicdiastereomer selected from the group of peptides consisting of thoseherein designated 94 and 95 (SEQ ID NO:94–95, respectively), of thesequences: 94) HN-Lys Leu Leu Leu Lys Leu Leu Leu Lys Leu Leu Lys-CO,and 95) HN-Lys Leu Leu Leu Lys Leu Lys Leu Lys Leu Leu Lys-CO.
 11. Thepeptide according to claim 2, wherein the net positive charge greaterthan +1 is due to the amino acid composition or to the addition ofpositively charged chemical groups, or which hydrophobicity is decreasedby the addition of polar amino acids selected from the group consistingof serine, threonine, methionine, asparagine, glutamine and cysteine.12. A peptide according to claim 1 consisting of a non-natural syntheticpeptide having from 6 to 12 amino acid residues or a non-naturalsynthetic cyclic peptide having from 6 to 14 amino acid residues and anet positive charge which is greater than +1, said peptide comprisingsolely hydrophobic amino acid residues excepting glycine and tyrosine,and positively charged and polar amino acid residues, wherein at leastone but not all of such amino acid residues is a D-amino acid, saidpeptide having a ratio of hydrophobic to positively charged amino acidssuch that the peptide is cytolytic to pathogenic cells but does notcause cytolysis or red blood cells, and having a sequence of amino acidssuch that the same amino acid sequence in which each residue is in theL-configuration is not found in nature, and cyclic derivatives thereoffrom 6 to 14 amino acid residues, with the proviso that said peptide isnot that of SEQ ID NO:1, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:23.13. The peptide according to claim 12 wherein the positively chargedamino acid is selected from the group consisting of lysine, arginine andhistidine, the hydrophobic amino acid is selected from the groupconsisting of leucine, isoleucine, alanine, valine, phenylalanine,proline, and tryptophan, and the polar amino acid is selected from thegroup consisting of serine, threonine, methionine, asparagine, glutamineand cysteine.
 14. The peptide according to claim 13 consisting of acyclic peptide in which the hydrophobic amino acid is leucine, thepositively charged amino acid is lysine, and the polar amino acid iscysteine; and said cyclic peptide is selected from the group of peptidesconsisting of those herein designated 92–93 (SEQ ID NOS:92–93,respectively), of the sequence: 92) Cyclic Cys Lys Leu Leu Leu Lys LeuLeu Leu Lys Leu Leu Lys Cys, 93) Cyclic Cys Lys Leu Leu Leu Lys Leu LysLeu Lys Leu Lys Cys.
 15. A peptide according to claim 1 being a mixtureof a plurality of peptide diastereomers, each peptide having at least 6amino acids and having a net positive charge which is greater than +1,said peptide comprising a hydrophobic L-amino acid, a positively chargedL-amino acid and a D-amino acid, said mixture being obtained by solidphase synthesis wherein at each coupling step a mixture composed of 1 eqof each of the amino acids is added to the reaction, followed by HFcleavage.
 16. A peptide mixture according to claim 15 wherein the aminoacids are L-lysine, L-leucine and D-leucine and the resulting mixturecontains 3¹² different 12-mer peptide diastereomers composed of L-Lys,L-Leu and D-Leu.
 17. A peptide according to claim 1 being a randomcopolymer having a net positive charge which is greater than 30 1, saidrandom copolymer consisting of a hydrophobic L-amino acid, a positivelycharged L-amino acid and a D-amino acid in a ratio of hydrophobic topositively charged amino acids such that the copolymer is cytolytic topathogenic cells but does not cause cytolysis of red blood cells. 18.The random copolymer according to claim 17, consisting of L-lysine,L-leucine and D-leucine in the molar ratio 1:1:1, 2:1:1 or 3:1:1.